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Cholesterol Interacts with Transmembrane α-Helices M1, M3, and M4 of the Torpedo Nicotinic Acetylcholine Receptor: Photolabeling Studies Using [ 3 H]Azicholesterol †
Abstract
The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.
... PLGICs are modulated by their lipid environment, but exactly where lipids bind, and how they act, is poorly understood [9][10][11]. Via reconstitution experiments, cholesterol (CHOL) and anionic lipids have been demonstrated to be crucial for nAChR function [12][13][14][15]. In pure phosphatidylcholine (PC) or phosphatidylethanolamine (PE) membranes, nAChRs bind agonist, but remain in an unresponsive, non-conductive state (that has been termed the uncoupled state). ...
... Since this is where ivermectin, a known positive allosteric modulator, has been shown to bind, it suggests that cholesterol might act as an intrinsic membrane modulator for receptor activation. This is supported by experimental evidence that cholesterol is crucial for the activation of the nicotinic acetylcholine receptor [6,12,13,15]. Overall, the different lipid types compete with each other for the same interaction regions on the receptor surface. Particularly, the neutral phospholipids PC, PE and SM can all interact with the same parts of the receptor. ...
... The nAChR is the best studied member of the pLGIC superfamily with regards to proteinlipid interactions [48]. Cholesterol and anionic lipids have both been shown to be essential for nAChR function [12][13][14][15]. nAChR reconstituted in pure PC or PE membranes bind agonist, but do not open. ...
Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor's conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.
... General anesthetics and other synthetic lipophilic compounds also bind to these same non-annular sites on both Torpedo and neuronal nAChRs [69][70][71]. On the other hand, a photoactivatable cholesterol analog only labels lipid exposed surfaces of the Torpedo M1, M3, and M4 TMD -helices (see below and Fig. 3) [72]. In addition, cholesteryl hemisuccinate covalently tethered to the glycerol backbone of PC is equally as effective as free cholesterol in supporting nAChR function in reconstituted PC/PA membranes [73]. ...
... A CRAC motif is located just prior to the transmembrane -helix, M1, in most subunits of the muscle type nAChR, proximal to the proposed M1-M2-M3 non-annular site noted above. An inverted or mirror image CRAC motif, termed CARC ((K/R)-X 1-5 -(Y/F)-X 1-5 -(L/V)), is also located on the M4-facing surface of M1 adjacent to a proposed annular cholesterol site [75][76][77], as well as on the γM4 -helix adjacent to a site of 3 H-azicholesterol labeling [72]. Both docking studies and molecular dynamics simulations suggest that these CARC motifs, along with other regions of the TMD, interact with cholesterol [77]. ...
... Interestingly, the cryo-EM structures of the detergent solubilized and saposin nanodisc-reconstituted 42 and 34 nAChRs solved in the presence of cholesteryl hemisuccinate exhibit distinct regions of electron density in the cytoplasmic leaflet at the periphery of the TMD (Fig. 3). This electron density is located on both sides of M4 at the M4/M1 and M4/M3 interfaces of each subunit, with the binding sites adjacent to residues at positions analogous to those covalently labeled in the Torpedo nAChR by a photoactivatable cholesterol probe [72]. Significantly, these densities disappear from the 34 nAChR cryo-EM structures when cholesteryl hemisuccinate is not included during sample preparation. ...
Pentameric ligand-gated ion channels (pLGICs) play a central role in synaptic communication and are implicated in a plethora of neurological disorders leading to human disease. Membrane lipids are known to modulate pLGIC function, but the mechanisms underlying their effects are poorly understood. Recent structures reveal sites for the binding of membrane lipids to pLGICs, thus providing a structural basis for interpreting functional data on pLGIC-lipid interactions. Here, we review the literature describing the known functional effects of membrane lipids on different members of the pLGIC superfamily and highlight pLGIC structures that exhibit bound lipids. We discuss new insight into the mechanisms of pLGIC-lipid interactions that has been derived from these recent structures.
... Molecular dynamics (MD) simulations also identify an intrasubunit binding site for cholesterol in nAchR (22). While these studies suggest cholesterol directly interacts with pLGICs, experimental evidence for the location of cholesterol binding sites in pLGICs is limited (23). ...
... Site identification was enhanced with MS1 doublets from FLI-tag, and stoichiometry was confirmed with intact protein MS. This data is consistent with previous studies that suggest cholesterol can bind to specific sites in pLGICs, such as GLIC (29), nAchR (22,23), and GABA A R (21). Molecular dynamic (MD) simulation studies of cholesterol binding to GLIC (29) or nAchR (22) identified a similar intrasubunit site; in the case of the nAchR, multiple binding orientations were identified, one of which is consistent with our photolabeling data in GLIC. ...
... Molecular dynamic (MD) simulation studies of cholesterol binding to GLIC (29) or nAchR (22) identified a similar intrasubunit site; in the case of the nAchR, multiple binding orientations were identified, one of which is consistent with our photolabeling data in GLIC. A PAL study of nAchR using [ 3 H] 6-azicholesterol identified labeled residues at the Nterminus (intracellular side) of TM4, as well as the C-terminus (extracellular side) of TM4 (23). These labeled residues do not delineate cholesterol binding pockets, but are consistent with the pockets we mapped in GLIC for an intersubunit and intrasubunit site, respectively. ...
Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3‑OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3‑OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.
... Molecular modeling simulations suggest that this domain displays a remarkable fit for cholesterol 8 , with a predicted energy of interaction of − 60 kJ.mol −1 . Interestingly, photolabeling studies using [ 3 H]Azicholesterol (Fig. 1A) have identified this region as an actual cholesterol-binding domain in the Torpedo AChR 18 . ...
... Indeed, the carboxylate group of Asp-448 was the atomic group closest to Azicholesterol. In particular, the lateral Azi-group did not interact with any of the apolar amino acid residues of TM4, in full agreement with the lack of radioactive labeling of these residues 18 . We studied next the interaction of natural cholesterol with the TM4 domain of Torpedo AChR γ -subunit (Fig. 1C). ...
... We have now subjected this to experimental test by studying the interaction of a prototype CARC domain with cholesterol monolayers. Our strategy for selecting such a representative CARC domain was based on photolabeling studies which identified Asp-448 as the main target for [ 3 H]Azicholesterol in the 4 th TM domain of the Torpedo AChR γ -subunit 18 . Since this residue is contiguous to a CARC domain, we decided to challenge our molecular dynamic simulation methods with this result. ...
Cholesterol controls the activity of a wide range of membrane receptors through specific interactions and identifying cholesterol recognition motifs is therefore critical for understanding signaling receptor function. The membrane-spanning domains of the paradigm neurotransmitter receptor for acetylcholine (AChR) display a series of cholesterol consensus domains (referred to as “CARC”). Here we use a combination of molecular modeling, lipid monolayer/mutational approaches and NMR spectroscopy to study the binding of cholesterol to a synthetic CARC peptide. The CARC-cholesterol interaction is of high affinity, lipid-specific, concentration-dependent, and sensitive to single-point mutations. The CARC motif is generally located in the outer membrane leaflet and its reverse sequence CRAC in the inner one. Their simultaneous presence within the same transmembrane domain obeys a “mirror code” controlling protein-cholesterol interactions in the outer and inner membrane leaflets. Deciphering this code enabled us to elaborate guidelines for the detection of cholesterol-binding motifs in any membrane protein. Several representative examples of neurotransmitter receptors and ABC transporters with the dual CARC/CRAC motifs are presented. The biological significance and potential clinical applications of the mirror code are discussed.
... Subsequent studies with photoactivatable sterols, which are not necessarily bona fide functional cholesterol substitutes, have provided further insight into putative regions of the AChR involved in interactions with cholesterol; the label was found in peptides that contain almost exclusively the a-TM4, a-TM1 and c-TM4 membrane spanning segments 8,9 . More recent photoaffinity labeling studies using azido-cholesterol analogs led to the identification of putative cholesterol-AChR interaction sites on the TM segments TM4, TM3, and TM1 of each subunit, with TM4 having the greatest interaction with membrane cholesterol, and in all cases fully overlapping the lipid-protein interface of the AChR 10 . ...
... It is also interesting to note that the CARC sequence satisfies several other criteria to qualify as a cholesterol binding motif: i) CARC is present in TM1, a receptor region firmly established as a membrane-spanning domain and, more importantly, to interact with membrane lipids 19 ; ii) there appears to be a good correlation between the amino acids in the CARC motifs in the AChR subunits and some of the residues in TM1 recently postulated by Brannigan et al. 20 to be involved in AChR-cholesterol interactions. iii) In addition, the Asp residue at the N-terminal region of the bTM4 segment, postulated by Hamouda et al. 10 to interact with cholesterol, is immediately adjacent to the CARC segment. ...
... A more recent proposal 20 is based on the observation of "gaps" in the electron density maps of the AChR cryoelectron microscopy images of Unwin and colleagues 21 : up to 5 cholesterol molecules could be accommodated into these deeply buried inter-subunit "holes", with a total of 15 cholesterol molecules per receptor molecule. Both Hamouda et al. 10 and our laboratory 24 had calculated the same number of cholesterol molecules from independent photolabeling and electron spin resonance experimental data, respectively, but unlike the deeply buried cholesterols postulated from in silico calculations in the more recent proposal 20 , all the cholesterol molecules readily exchange with bulk lipids 10,24 ( Supplementary Fig. S6 online). ...
We report the finding of a new putative cholesterol-recognition motif in the transmembrane region of membrane proteins like the nicotinic acetylcholine receptor (AChR) and other members of the Cys-loop superfamily of neurotransmitters involved in rapid synaptic transmission, as well as in members of the important superfamily of G protein coupled receptors (GPCRs) involved in Alzheimer's disease. The motif is an inverted CRAC domain (cholesterol recognition/interaction amino acid consensus) which we have named CARC and is present in the transmembrane regions of the receptor subunits having extensive contact with the surrounding lipid, predominantly in the first (TM1) segment in the case of the AChR, and optimally suited to convey cholesterol-mediated signaling. Three cholesterol molecules could be docked on the transmembrane regions of the five human AChR subunits, rendering a total of 15 cholesterol molecules per AChR molecule, adding up to a total of about 200 kJ.mol-1 per receptor molecule, i.e. ∼40% of the total lipid solvation free energies for the whole AChR molecule. The novel motif is remarkably conserved among Cys-loop receptors along the phylogenetic scale, from prokaryotes to humans, its high degree of conservation suggesting that it could be responsible for some of the structural/functional properties of these transduction macromolecules.Reference: Baier, C.J., Fantini, J. and Barrantes, F.J. (2011). Sci. Rep. 1:00069.
... Both the properties and the characteristics of the membrane where it is embedded are essential for its function as well as for its biosynthesis and correct assembly [21][22][23][24][25]. At the same time, the nAChR influences its nearby lipids [6,[25][26][27][28][29][30][31]. A layer of immobilized lipids with different characteristics from those of bulk lipids encircles the muscle nAChR [26] where two different populations of lipids, namely non-annular and annular lipids [27], can be distinguished. ...
... Although the effect of the increment of Chol on the membrane order reaches a saturation state, it has been reported that it influences nAChR behavior. A Chol fraction of 33% in the membrane was reported as the optimum for nAChR activity [31,88]. An inhibition of ~52% of the macroscopic currents of nAChRs from T. californica was observed in Xenopus laevis oocytes when the Chol/phospholipid ratio was increased from 0.51 to 0.87 [89]. ...
Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR.
Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.
... The effects of cholesterol on nAChR stability and functionality have been studied by reconstitution of nAChR in model lipid bilayer at different mole fractions. The effect of cholesterol on the nAChR functionality has been extensively studied and reported [56,[71][72][73][74][75][76][77][78][79][80]. ...
... Despite the increase in the number of X-Ray determined membrane protein structures, which showed resolved lipids such as cholesterol, there are not enough studies to correlate function with the presence of these lipids [115][116][117][118][119]. Furthermore, an overview of 73 crystallographic structures with cholesterol-bound of soluble and membrane proteins have shed light into the structural characteristics of cholesterol binding, where in the majority of the cases studied, cholesterol is positioned with its α-face oriented toward the β-strands and its β-face facing the α-helical structure [120]. CARC motifs have been identified in the Torpedo californica nAChR [77,105,121,122]. Experimental evidence supports the hypothesis that cholesterol induces amyloid-beta aggregation by increasing β-sheet formation and aromatic side chain mutation eliminate the cholesterol aggregation effect [123,124]. ...
Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification.
We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of β-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-β-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-β-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.
... The temporal quality of neuronal receptor allostery typically poses a challenge for experimental characterization as some allosteric states exist transiently, however the approach of using photocrosslinking provides an opportunity to probe the dynamics of receptor gating and desensitization 50,51 . Photoaffinity labeling studies identified the propofol-binding site of GLIC 52 and GABAR 53 as well as cholesterol interactions with the peripheral-type benzodiazepine receptor 54 , nAChR 55,56 , and GlyR 49 . The incorporation of sensitive massspectrometry (MS)-based approaches to sensitively identify sites of photocrosslinking has the potential to identify allosteric dynamics of protein structure in membranes and to examine the role of lipids in receptor allostery 49,51 . ...
... Taken together, state-dependent crosslinking of M4 suggests a outward twisting motion as the helix allosteric transitions which is consistent with general TMD movements observed between cryo-EM GlyR structures 71 . Cholesterol crosslinking within the portion of M4 nearing the M3-M4 loop (bilayer lower leaflet region) is consistent with similar studies of nAChR 56 showing N-terminal M4 cholesterol crosslinking. Our state-dependent cholesterol CX-MS study expands upon the lipid-channel studies by being able to not only highlight the specific crosslinking locations throughout the entire receptor, yet also distinguish the differential crosslinks in a state-dependent manner. ...
Pentameric ligand-gated ion channel (pLGIC) allostery is dependent on dynamic associations with its diverse environment. The cellular membrane’s lipid composition influences channel function with cholesterol being a key regulator of channel activity. Human α 1 glycine receptor (GlyR) was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at physiological cholesterol:lipid ratios with aliquots of azi-cholesterol, a photoactivatable non-specific crosslinker. The receptor in vesicles was then enriched in either a resting, open, or desensitized state prior to photocrosslinking. Following photoactivation, crosslinked cholesterol-GlyR was trypsinized and sites of direct covalent attachment to peptides were identified by targeted MS/MS. Dozens of state-dependent crosslinks were identified and differential patterns of cholesterol-GlyR crosslinks were observed in the extracellular region nearing the lipid bilayer, in the M4 transmembrane helix, and in the large intracellular M3-M4 loop. Unique crosslinks in comparative studies identify changes in lipid accessibility or modulation of hydrophobic cavities in GlyR as a function of receptor allostery. Most notably, the outward twisting of M4 and differential crosslinking within the M3-M4 loop provide new insight into allosteric repositioning of GlyR. More generally, this study provides an accurate and sensitive approach to mapping the protein-lipid interactions to discern state-dependent structural movements of membrane proteins embedded in lipid-bilayers.
Significance
Ion channels are highly allosteric molecular machines whose structure and function are sensitive to lipids and ligands. While the structures of many pLGICs are known, these are often truncated forms of the receptor in a membrane-mimetic environment locked in ligand-bound conformational states that may not accurately reflect the conformation and dynamics of the receptor in a native lipid environment. Crosslinking coupled with mass spectrometry (CX-MS) has the capability of interrogating the structure of full-length receptors in a lipid environment. In this study, CX-MS was used to identify state-dependent cholesterol-GlyR interactions to identify differential cholesterol accessibility as a function of channel dynamics upon gating and desensitization.
... For example, cholesterol homologs were used to map sites of cholesterol interactions with the peripheral-type benzodiazepine receptor providing for the identification of putative consensus sites for cholesterol binding (26). Cholesterol photoaffinity probes have also characterized lipid-protein interactions with ion channels (27,28). The advent of mass spectrometry (MS) methodologies offers the opportunity to examine membrane protein structure (29) to more sensitively and accurately detect and map the interaction of lipids with membrane proteins (30)(31)(32). ...
... Photoactivatable cholesterol analogs have been utilized to efficiently probe the lipidaccessible regions of transmembrane protein (27). The cholesterol analog used in this study includes a photoactivatable diarizine moiety at carbon seven (Fig. 1). ...
Altered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipid-accessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino- and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility.
... Recent studies revealed many limitations of the application of chiral analogues. As described for nAchR, despite the same effects of cholesterol and its chiral analogues on protein function, a direct interaction has been proposed between nAchR and cholesterol/chiral analogues mediated by a "lax cholesterol binding site" [219,220] (see later in chapter 5.4.). In that way the lack of stereospecificity cannot exclude the existence of direct protein-cholesterol interactions as suggested before, thus the application of stereoisomers is more sensitive to discriminate between the direct and indirect interactions if cholesterol and its isomers have different or opposite effects on protein functions, which indicates a probable direct cholesterol-protein interaction [2,49]. ...
... Cholesterol has long been suggested as an important regulator of nAChR function, since its presence was required for transport activity when reconstituted into vesicles [349]. Direct J o u r n a l P r e -p r o o f cholesterol binding to M4, M1 and M3 segments of all channel subunits was initially proposed based on photoactivatable sterol probe incorporation [220] and MD simulations demonstrating three binding sites per subunit [134]. Consistently, CARC and CARC-like motifs were described in membrane embedded regions, rendering a total of 15 cholesterols bound per nAChR [124]. ...
As described in the literature the interaction between cholesterol and membrane proteins can occur via direct, ligand-like and indirect mechanisms, in which cholesterol effects are mediated by alterations in the biophysical properties or in the protein-organizing functions of the lipid membrane. Early studies emphasized the importance of indirect and raft-mediated effects, but improvements in computational and structural imaging techniques allowed the definition of a wide range of functionally active cholesterol binding domains and sites suggesting the relevance of direct cholesterol effects in various proteins. However, the intramolecular rearrangements induced by cholesterol leading to modulation of ion channel gating, membrane transport and receptor functions still have not been revealed. In this review we summarize the novel findings of the topic by focusing on recent studies about direct and indirect effects of cholesterol on potassium ion channels, and we extend the review to transporters and receptors with different domain structures to introduce the general mechanisms of cholesterol action among membrane proteins. We propose that rather than pure direct or indirect effects, cholesterol action on membrane proteins can be better described as a mixture of indirect and direct interactions with system-specific variability in their contributions, which can be explored by using a multi-level approach employing multiple experimental techniques.
... Meanwhile, in all α-subunits from cation-selective receptor types, a bulky hydrophobic residue (F, I, L or M) is conserved at this position. Our assignment of this cholesterol binding site is consistent with mapping studies using photo-activatable cholesterol analogues 26,27 . ...
... Uniprot accession IDs are provided 40 . Residues mapped using a photoreactive cholesterol analogue in Torpedo californica (TC P02710) 26,27 are highlighted in cyan. All other sequences are from Homo sapiens (HS P43681, P11230, P17787, Q05901, P30926 for α4 and β1-4, respectively). ...
Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries 1 . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction2,3 and congenital epilepsy4,5. Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to 'break' symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.
... Cholesterol analogs used in early photoaffinity labeling experiments have either restricted the characterization of labeling to the intact subunit level (Middlemas and Raftery, 1987;Fernandez et al., 1993) and/or employed photoactivatable sterols that are likely not functional cholesterol substitutes (Corbin et al., 1998b;Blanton et al., 1999). More recent photoaffinity studies led to the identification of putative cholesterol-AChR interaction sites at the TM4, TM3, and TM1 segments of each subunit, fully overlapping the lipid-protein interface of the AChR (Hamouda et al., 2006). The TM4 segment showed the greatest interaction with cholesterol. ...
... For αTM4, the labeling pattern was consistent with azicholesterol incorporation into αGlu-398, αAsp-407, and αCys-412, that is, a rather shallow region in the NH-term of the TM4 segment. Hamouda et al. (2006) remarked the striking labeling of a conserved Asp at the N-terminus of each TM4 segment , together with the only acidic side chain at the C-terminus of the TM4 segments (βAsp-457). ...
Introduction The AChR is Surrounded by Lipids in the Liquid-Ordered Phase Stoichiometry and Selectivity of the Protein-Vicinal Lipid Influence of Cholesterol on AChR Secondary Structure Influence of Cholesterol on Agonist-Induced Conformational States and Ion Translocation Function The Common “Three-Ring” Scaffold in Cys-Loop Receptor Transmembrane Topology: From Bacterial Homologs to Eukaryotes Cholesterol Binding to Nonannular Sites on the AChR Molecule A New Cholesterol-Recognition Motif in the AChR is also Present in GPCRs Acknowledgments References
... Inward and outward currents were similarly reduced (Fig 10), which suggests that the binding site of the pancuronium is apart from the ionic pathway of nAChR. Radioactively labeled cholesterol binds to the transmembrane domain proximal to the intracellular domain of the muscle-type nAChR [32]. Combined with our results, aminosteroids may bind to the transmembrane domain and/or interface between the transmembrane and intracellular domain and exert the inhibitory effect. ...
Muscle relaxants are indispensable for surgical anesthesia. Early studies suggested that a classical non-depolarizing muscle relaxant pancuronium competitively binds to the ligand binding site to block nicotinic acetylcholine receptors (nAChR). Our group recently showed that nAChR which has two distinct subunit combinations are expressed in zebrafish muscles, αβδε and αβδ, for which potencies of pancuronium are different. Taking advantage of the distinct potencies, we generated chimeras between two types of nAChRs and found that the extracellular ACh binding site is not associated with the pancuronium sensitivity. Furthermore, application of either 2 μM or 100 μM ACh in native αβδε or αβδ subunits yielded similar IC 50 of pancuronium. These data suggest that pancuronium allosterically inhibits the activity of zebrafish nAChRs.
... This individual behavior translates into phospholipid/cholesterol ratios for CF-4 and CF-6 of approximately 5 and 7, which are similar to those reported for lipid-like detergent analog mentioned above (see Fig. 2). Studies carried out in the late 1980s where the effect of lipid composition on the functionality of the solubilized Tc-nAChR was determined suggested that at least a ratio of 45 lipids/nAChR should be present to observe activity (Marsh et al. 2002;Hamouda et al. 2006a). Furthermore, the amount of cholesterol present in model membranes that support nAChR functionality should be approximately 35 mol%, since this value is similar to that found in the native membranes Tc-nAChR-DC (Marsh et al. 2002). ...
The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (− 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.
Graphical abstract
... properties (Kapoor et al., 2019). Structural studies and photolinking have also shown various lipids bound to, and in some cases affecting the function of, the GABAAR, GlyR, GluCl and GLIC (Althoff et al., 2014;Budelier et al., 2019;Cheng et al., 2018;Hamouda et al., 2005;Hibbs and Gouaux, 2011;Huang et al., 2017;Laverty et al., 2019;Tong et al., 2019). ...
Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the human nervous system, and contribute to a range of muscle, gut, and neurological functions. Elucidating their mechanism of action and how it might be modulated would improve our understanding of the nervous system, and contribute to building tools to treat diseases arising from dysregulated pLGICs. The outermost lipid-facing transmembrane helix (M4) and the lipids surrounding it have recently emerged as important factors in pLGIC function. To investigate the role of the M4 helix in cation-selective mammalian pLGICs, I studied the effects of mutations in the M4 helices of the 5-HT3A and α4β2 nACh receptors. I used a membrane potential-sensitive fluorescent dye, two-electrode voltage clamp and manual patch-clamp for functional characterisation of mutant receptors in HEK293 cells and Xenopus oocytes, and radioligand binding and immunofluorescence to assess ligand binding and receptor expression. I show that 1 out of 28 alanine mutations in the 5-HT3AR M4 and 8 out of28 double alanine mutations in the α4β2 nAChR abolish receptor function in HEK cells without ablating ligand binding, indicating that the M4 helices of these cation-selective pLGICs are involved in, and can modulate, receptor function. I explored the mechanism of action of these key M4 residues by characterising prospective interaction partners, and identified a potential chain of interactions going from the outermost M4 helix all the way to the channel pore. I also show that eight of the nine 5-HT3AR and α4β2 nAChR mutants that showed ligand binding but no receptor function in HEK cells, showed WT-like function when expressed in Xenopus oocytes. In addition, M4 mutations that altered the function of receptors expressed in HEK cells had different effects on receptors expressed in oocytes. Together this shows that the role of the M4 helix in cation-selective pLGIC function depends on the expression system. For comparison, I investigated another peripheral helix in the 5-HT3AR; the N-terminal helix, which rests above the extracellular domain of mammalian pLGICs, and showed that it is important for correct receptor expression. Overall, this work shows that the M4 helix of cation-selective pLGICs is an attractive target for receptor modulation by small-molecule binding, as this helix is both accessible, poorly conserved between pLGICs, and intimately involved in receptor function. It has also laid the groundwork for further understanding the functional mechanism of pLGICs, especially the interactions of the M4 helices and with the rest of the transmembrane helical bundle. Finally, it has highlighted the dependency on the expression system of both pLGIC function and of the role of the M4 helix, and emphasises the need to understand the native environment of these receptors and how that modulates function.
... Each structure exhibits regions of electron density at the periphery of the TMD that was modeled as cholesterol ( Figure 2). The bound cholesterol, located in the cytoplasmic leaflet at both the M4-M1 and the M4-M3 interfaces of each subunit, is close to residues covalently labeled in the Torpedo nAChR by a photoactivatable cholesterol probe [56]. Notably, the electron density attributed to cholesterol disappears when the cryo-EM samples are prepared in the absence of CHS. ...
Pentameric ligand-gated ion channels (pLGICs) play a leading role in synaptic communication, are implicated in a variety of neurological processes, and are important targets for the treatment of neurological and neuromuscular disorders. Endogenous lipids and lipophilic compounds are potent modulators of pLGIC function and may help shape synaptic communication. Increasing structural and biophysical data reveal sites for lipid binding to pLGICs. Here, we update our evolving understanding of pLGIC–lipid interactions highlighting newly identified modes of lipid binding along with the mechanistic understanding derived from the new structural data.
... Cholesterol is a waxy like substances and it is hydrophobic in nature. All cellular cholesterols are distributed unlikely inside the membrane from N-C terminus and to identify cholesterol binding sites of all motifs among seven helices named as helix 1 to helix 7 which is shown if figure 3 below [19][20][21][22]. Most of the researchers focused their work only on plasma membrane receptor like GPCR. ...
The researches have been made on G-protein coupled receptors (GPCRs) over the long-ago decades. GPCR is also named as 7-transmembrane (7TM) receptor. According to biological prospective GPCRs consist of large protein family with respective subfamilies and are mediated by different physiological phenomena like taste, smell, vision etc. The main functionality of these 7TM receptors is signal transduction among various cells. In human genome, cell membrane plays significant role. All cells are made up of trillion of cells and have dissimilar functionality. Cell membrane composed of different components. GPCRs are reported to be modulated by membrane cholesterol by interacting with cholesterol recognition amino acid consensus (L/V-X (1-5) -Y-X (1-5) -R/K) (CRAC) or reverse orientation of CRAC (R/K-X (1-5) -Y-X (1-5) -L/V) (CARC) motifs present in the TM helices. Among all, cholesterol is one who is regulated by membrane proteins. Here we took GPCR as membrane proteins and this protein modulates membrane cholesterol. According to cell biology, GPCR regulates a wide diversity of vital cellular processes and are targeted by a huge fraction of approved drugs. In this paper we have concentrated our investigation on membrane protein with membrane cholesterol. A hybrid algorithm consisting of spectral clustering and support vector machine is proposed for prediction of membrane cholesterol with GPCR. Spectral clustering uses graph nodes for calculating the cluster points and also it considers other concept such as similarity matrix, low-dimensional space for projecting the data points and upon this parameter at last construct the cluster centre. Supervised learning method is used for solving regression and classification problems. From the analysis we found that our result shows better prediction accuracy in terms of time complexity when compared with two existing models such as fuzzy c-means (FCM) and rough set with FCM model.
... Support vector machine (SVM) plays a significant role for prediction and classification among dissimilar objects. For that purpose we have developed a hybrid approach K-means with support vector machine for prediction of membrane protein GPCR with membrane cholesterol [17][18][19][20]. Here we have explored a hybrid approach based on K-means and support vector machine. ...
In cell physiology, eukaryotic membrane components have played significant role in every aspect of human body. Amid all, membrane cholesterol is one who has the power to drop off the permeability of biological membranes to a variety of solutes. Generally, cholesterol is distributed heterogeneously in membrane protein. Additionally, the length of membrane protein is laid from extracellular region to intracellular region, and membrane cholesterol is always targeted in both forward and backward direction. We know that G-protein-coupled receptor is a superfamily in mammalian cells and it contains more than 800 proteins in it. Among these 800 proteins, half of them are olfactory proteins. So it is very difficult to predict exact signature motif of cholesterol with GPCR protein. So we have focused our research except on olfactory receptor. In case of data mining applications, clustering plays a crucial role. Clustering is nothing but grouping of similar objects in one cluster points. There are numerous algorithms which have been utilized for clustering the same data points. Among all K-means is an important algorithm which is used when we have unlabeled data. Support vector machine (SVM) plays a significant role for prediction and classification among dissimilar objects. For that purpose we have developed a hybrid approach, that is, K-means and support vector machine, for prediction of membrane protein GPCR and membrane cholesterol, and this experiment provides enhanced prediction results according with our data.
... The microdomains are apparently stabilized by properties of the protein surfaces and by the high cholesterol content of the postsynaptic membrane. The TM helix M4, which has an affinity for cholesterol [11] and tilts into the lipids away from the body of the receptor, is a stabilizing influence in the outer leaflet. The sub-membrane helix MX, which sterically excludes the large phospholipid headgroups from the vicinity of the TM helices, is a stabilising influence inner leaflet. ...
Many new structures of membrane proteins have been determined over the last decade, yet the nature of protein-lipid interplay has received scant attention. The postsynaptic membrane of the neuromuscular junction and Torpedo electrocytes has a regular architecture, opening an opportunity to illuminate how proteins and lipids act together in a native membrane setting. Cryo-EM images show that cholesterol segregates preferentially around the constituent ion channel, the nicotinic acetylcholine receptor, interacting with specific sites in both leaflets of the bilayer. In addition to maintaining the transmembrane α-helical architecture, cholesterol forms microdomains - bridges of rigid sterol groups that link one channel to the next. This article discusses the whole protein-lipid organisation of the cholinergic postsynaptic membrane, its physiological implications, and how the observed details relate to our current concept of membrane structure. I suggest that cooperative interactions, facilitated by the regular protein-lipid arrangement, help to spread channel activation into regions distant from the sites of neurotransmitter release, thereby enhancing the postsynaptic response.
... The shape and size of the former are suggestive of cholesterol (Fig. 5b). Indeed, M4 is a well-documented sterol binding site in nAChR 13,33 . Cholesterol occupied similar positions in the apo-and serotonin-5HT 3 R-Salipro structures, nested in a hydrophobic pocket between pre-M1, M1, post-M4, and the Cys loop (Fig. 5b, c). ...
Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT 3A serotonin receptor (5HT 3 R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT 3 R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, 'coupled' state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT 3 R by the membrane environment, and a model for asym-metric activation of the receptor.
... The original CRAC motif was discovered in the cytosolic portion of a cholesterol transport protein, the Peripheral-type benzodiazepine receptor (52). A CRAC inverted motif N-(K/R)X 1-5 (Y/F)X 1-5 (L/V)-C, defined as CARC motif, was studied in the integral membrane protein human Nicotinic acetylcholine receptor by molecular docking experiments after the cholesterol interaction to single helices in the receptor had been tested earlier (53,58) (see Figure 1. 3 B). Both the CRAC and the CARC motif are defined by a triad of basic (R/K), aromatic (F/Y) and aliphatic (L/V) amino acid residue (54). ...
The Metabotropic glutamate receptor 2 (mGluR2) belongs to the family of G-protein coupled receptors, a specific class of transmembrane proteins involved in cellular signaling. The functionality of such transmembrane proteins has been identified to largely depend on their microenvironment, namely the lipid bilayer surrounding them. However, the regulation of the receptors by their lipid microenvironment remains poorly understood. In particular, it remains unclear how specific protein-lipid interactions may modulate the function of mGluR2. In the last years, general motifs for non-covalent cholesterol and sphingolipid interaction within helical domains of transmembrane proteins have been described. In these motifs, both tryptophan and tyrosine residues have been found to play a decisive role. For that reason, an alanine mutagenesis screening, targeting tryptophan and tyrosine residues at the transmembrane surface, was carried out in the search of specific sphingolipid or cholesterol interaction sites for mGluR2. For the different Y→A and W→A variants, surface biotinylation and co-immunoprecipitation showed that neither trafficking nor dimerization were disturbed by substitution of these aromatic residues. In contrast, cellular photo-crosslinking assays demonstrated that cholesterol binding was compromised if one tyrosine residue located at the helix five or another at the helix six was replaced. Thus, these experiments suggested these two helices to contain specific cholesterol binding sites. To get a better molecular insight into these specific protein-lipid interactions, lipid binding to the transmembrane domain of mGluR2 was investigated in molecular dynamics (MD) simulation. The molecular dynamics simulations in GROMACS were performed in collaboration with the Max Planck tandem group of Dr. Camilo Aponte-Santamaría. All-atom and coarse-grained MD simulations of the mGluR2 transmembrane domain confirmed the experimental observation, by revealing a highly-localized density of cholesterol near these residues in helices five and six, which smeared out when they were changed to alanine in silico. The simulations also revealed flexibility of the protein structure at the exoplasmic end of helix six which changed upon introduction of point mutations. Overall, the work combining functional assays and MD simulations demonstrated the existence of specific cholesterol binding sites in mGluR2. It will be highly interesting to investigate the functional implications of this newly-found specific protein–cholesterol interaction on the activity and conformation of the receptor.
... As a non-annular lipid, the occurrence of allosteric binding sites is postulated ( Addona et al., 1998). It was suggested that the binding domain for Chol is at the nAChR lipid-protein interface, taking contact with the transmembrane subunits αM4, αM1, and γM4 ( Corbin et al., 1998); other studies identified interactions of Chol with the transmembrane segments M1, M3, and M4 of each subunit (Hamouda et al., 2006). By fluorescence quenching and energy-transfer measurements of T. californica reconstituted membranes, sites accessible to Chol but not to phospholipids were identified . ...
Biological membranes show lateral and transverse asymmetric lipid distribution. Cholesterol (Chol) localizes in both hemilayers, but in the external one it is mostly condensed in lipid-ordered microdomains (raft domains), together with saturated phosphatidyl lipids and sphingolipids (including sphingomyelin and glycosphingolipids). Membrane asymmetries induce special membrane biophysical properties and behave as signals for several physiological and/or pathological processes. Alzheimer’s disease (AD) is associated with a perturbation in different membrane properties. Amyloid-β (Aβ) plaques and neurofibrillary tangles of tau protein together with neuroinflammation and neurodegeneration are the most characteristic cellular changes observed in this disease. The extracellular presence of Aβ peptides forming senile plaques, together with soluble oligomeric species of Aβ, are considered the major cause of the synaptic dysfunction of AD. The association between Aβ peptide and membrane lipids has been extensively studied. It has been postulated that Chol content and Chol distribution condition Aβ production and posterior accumulation in membranes and, hence, cell dysfunction. Several lines of evidence suggest that Aβ partitions in the cell membrane accumulate mostly in raft domains, the site where the cleavage of the precursor AβPP by β- and γ- secretase is also thought to occur. The main consequence of the pathogenesis of AD is the disruption of the cholinergic pathways in the cerebral cortex and in the basal forebrain. In parallel, the nicotinic acetylcholine receptor has been extensively linked to membrane properties. Since its transmembrane domain exhibits extensive contacts with the surrounding lipids, the acetylcholine receptor function is conditioned by its lipid microenvironment. The nicotinic acetylcholine receptor is present in high-density clusters in the cell membrane where it localizes mainly in lipid-ordered domains. Perturbations of sphingomyelin or cholesterol composition alter acetylcholine receptor location. Therefore, Aβ processing, Aβ partitioning, and acetylcholine receptor location and function can be manipulated by changes in membrane lipid biophysics. Understanding these mechanisms should provide insights into new therapeutic strategies for prevention and/or treatment of AD. Here, we discuss the implications of lipid-protein interactions at the cell membrane level in AD.
... Photolabeling studies have shown that sterols interact directly with the nAChR's lipid-exposed TM helices. 329 Brannigan et al. demonstrated the presence of internal cholesterol binding sites in the cryo-EM structures of the nAChr channel. 330 Also, the authors compared the stability of these structures with and without cholesterol by atomistic MD simulations. ...
Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions.
... Neuronal nAChRs also are expressed in numerous other cell types and tissues, including endothelial cells, gastrointestinal tissues, glia, immune cells, keratinocytes, urinary bladder tissues, reproductive organs, and respiratory tissues [85][86][87][88][89][90][91][92][93][94][95][96]. In addition to the acetylcholine binding sites [20,40] other binding sites on nAChRs, such as the cholesterol-binding sites in M1-M4 segments [74,97] the α-neurotoxin-binding sites in the central loop C of the α subunit, [98] and binding sites for methyllycaconitine and synthetic antagonists [99] have been identified. As a result of their roles in the propagation of action potentials, cognitive function, and diverse central nervous system pathologies, they are hypothetical targets for many drugs and toxins [84,100]. ...
Background:
Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop ligandgated ion-channel (LGIC) superfamily, which also includes the GABA, glycine, and serotonin receptors. Many nAChR subunits have been identified and shown to be involved in signal transduction on binding to them of either the neurotransmitter acetylcholine or exogenous ligands such as nicotine. The nAChRs are pentameric assemblies of homologous subunits surrounding a central pore that gates cation flux, and they are expressed at neuromuscular junctions throughout the nervous system.
Methods and results:
Because different nAChR subunits assemble into a variety of pharmacologically distinct receptor subtypes, and different nAChRs are implicated in various physiological functions and pathophysiological conditions, nAChRs represent potential molecular targets for drug addiction and medical therapeutic research. This review intends to provide insights into recent advances in nAChR signaling, considering the subtypes and subunits of nAChRs and their roles in nicotinic cholinergic systems, including structure, diversity, functional allosteric modulation, targeted knockout mutations, and rare variations of specific subunits, and the potency and functional effects of mutations by focusing on their effects on nicotine addiction (NA) and smoking cessation (SC). Furthermore, we review the possible mechanisms of action of nAChRs in NA and SC based on our current knowledge.
Conclusion:
Understanding these cellular and molecular mechanisms will lead to better translational and therapeutic operations and outcomes for the prevention and treatment of NA and other drug addictions, as well as chronic diseases, such as Alzheimer's and Parkinson's. Finally, we put forward some suggestions and recommendations for therapy and treatment of NA and other chronic diseases.
... In contrast, it was concluded that nAChR is regulated by the direct interaction with the cholesterol molecule and that this interaction is not stereospecific. Notably, direct interaction of cholesterol with nAChR was further verified using a photoactivatable cholesterol analogue that cross-links with the protein upon UV illumination (Corbin, Wang, & Blanton, 1998;Hamouda, Chiara, Sauls, Cohen, & Blanton, 2006). It is also important to note, however, that the study of Baenziger and colleagues showed recently that nAChR is also regulated by the thickness of the lipid bilayer, indicating that both specific sterol-protein interactions and physical properties of the membrane are important for activating nAChR (daCosta et al., 2013). ...
Numerous ion channels have been shown to be regulated by the level of membrane cholesterol, but the mechanisms responsible for these effects are still not well understood. The key question in the field is how to discriminate between the contributions of the two central mechanisms that might be responsible for the sensitivity of ion channels to cholesterol: specific sterol–protein interactions or regulation of channels by the bilayer physical properties. Comparative analysis of cholesterol and its isomers on the function of an ion channel is a powerful tool to achieve this goal. An increasing number of studies show that cholesterol regulates several types of ion channels in a stereospecific manner, suggesting an involvement of specific sterol–protein interactions. However in this chapter, we present evidence that the stereospecificity of cholesterol–ion channel interactions may be mediated, not by a lack of binding, as has been generally assumed, but by the specificity of the interaction, which results in a functional effect, in the case of native cholesterol, and a lack of functional effect, in the case of a cholesterol isomer. In other words, accumulating evidence suggests that the structural requirements of ion channel cholesterol-binding sites are lax, allowing chiral isomers of cholesterol to bind to the same site in a nonstereospecific way, but the ability of a sterol to confer a functional effect on the channel activity can still be stereospecific. This is an important distinction both conceptually and methodologically. Indeed, our analysis shows that the orientations of cholesterol and its chiral isomer ent-cholesterol within a hydrophobic binding pocket of Kir2.2 are significantly different, and we propose that this difference may underlie distinct functional outcomes.
... In a recent work the working hypothesis was subjected to experimental test by studying the interaction of a prototype CARC domain with cholesterol employing lipid monolayer strategies and nuclear magnetic resonance (NMR) spectroscopy. Selection of a representative CARC domain for these experimental validations was based on previous photolabeling studies of the Torpedo nAChR with the cholesterol analogue probe [ 3 H]azicholesterol, which led to the identification of a (predominant) cholesterol-binding domain in the fourth transmembrane domain (TM4) of the human nAChR γ subunit (Hamouda, Chiara, Sauls, Cohen, & Blanton, 2006). Subsequent in silico computational approaches (Baier et al., 2011) led us to identify a typical CARC motif: 455-RVCFLAML-462 (the characteristic Arg, Phe, and Leu amino acid residues outlined bold and underlined) in the human γTM4 that incorporated most of the label in the photoaffinity studies. ...
Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined “CARC”). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC–CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif–protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.
Relevance of CARC and CRAC Cholesterol-Recognition Motifs in the Nicotinic Acetylcholine Receptor and Other Membrane-Bound Receptors. Available from: https://www.researchgate.net/publication/318333351_Relevance_of_CARC_and_CRAC_Cholesterol-Recognition_Motifs_in_the_Nicotinic_Acetylcholine_Receptor_and_Other_Membrane-Bound_Receptors [accessed Aug 1, 2017].
... We have recently challenged the hypothesis and submitted it to experimental test using two biophysical techniques: the interaction of a prototype CARC domain with cholesterol was studied using lipid monolayer techniques and nuclear magnetic resonance (NMR) spectroscopy. A representative CARC domain for these experimental corroborations was chosen on the basis of previous photolabeling studies of the Torpedo nAChR with the cholesterol analogue probe [ 3 H]azicholesterol, which led to the identification of a (predominant) cholesterol-binding domain in the 4th transmembrane domain (TM4) of the human nAChR γ subunit [62]. As mentioned in preceding paragraphs, our in silico computational approaches [8] led us to identify a typical CARC motif: 455-RVCFLAML-462 (the characteristic Arg, Phe, and Leu amino acid residues outlined bold and underlined) in the human γ TM4 that incorporated most of the label in the photoaffinity studies. ...
Once the sterol biosynthetic machinery had progressed over the course of several million years to yield cholesterol, this neutral lipid became an omnipresent and essential component of biomembranes in Eukaryotes. The hopanoids in Prokaryotes and eukaryotic sterols share the ability to provide stability and domain compartmentalization in membranes. Even more important is the intimate association of cholesterol with a wide range of cell-surface membrane proteins, probably responsible for its modulatory effects on neurotransmitter and hormone receptors and ion channels. These effects appear to be exerted via the membrane-embedded segments of essentially all members of the pentameric ligand-gated ion channel and G-protein-coupled receptor superfamilies, which possess consensus linear arrays of amino acid residues recognizing cholesterol with relatively
high affinity and specificity, an early evolutionary acquisition already present in ancient bacteria, conserved, and further improved
in Eukaryotes. This review focuses on the long-term relationship between cholesterol and these functionally important membrane protein superfamilies, and the ability of cholesterol to induce lateral segregation and ordered domain formation at the nanoscale in cell membranes.
... This is in good agreement with experimental results which suggested that the presence of integrins affects the cholesterol distribution (Pankov et al. 2005). Indeed, cholesterol has been suggested to interact with and possibly regulate a number of types of receptor (Hamouda et al. 2005;Hanson et al. 2008;Lingwood et al. 2011;Sahu et al. 2012;Shinoda et al. 2009). Our simulations confirm the electrostatic nature of the talin/bilayer interactions. ...
Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2–F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00232-016-9908-z) contains supplementary material, which is available to authorized users.
... A decade later photoactivatable cholesterol analogues were used to dissect subunit labeling into individual TM segment labeling [83,84]. The most recent work along these lines [85] confirmed the occurrence of sterol-recognition sites in the TM4, TM3, and TM1 segments of each subunit, fully overlapping the lipid-exposed rings of the nAChR. The TM4 segment showed the most extensive labeling with the cholesterol analogs. ...
The nicotinic acetylcholine receptor (AChR) is an integral membrane protein that transduces the binding of acetylcholine into a local depolarization of the postsynaptic membrane through the transient opening of a cation-selective channel. The AChR is a glycoprotein of about a quarter of a million molecular weight and is by far the best known of all neurotransmitter receptors from both a functional and a structural point of view. This is because the AChR macromolecule is very abundant in tissues such as the electric organ of fish, thus providing an excellent biological source for biochemical studies, and because of the availability of appropriate chemical tools such as α-toxins, which are quasiirreversible competitive antagonists of AChR function. These toxins have extremely high affinity for the receptor and have not only enabled the characterization of some pharmacological properties of the latter but have helped, among other things, to localize the protein in cells and tissues, to study state transitions elicited by agonists, and to purify the AChR by affinity chromatography (see reviews by Barrantes, 1983; Changeux et al., 1983).
... A decade later photoactivatable cholesterol analogues were used to dissect subunit labeling into individual TM segment labeling [83,84]. The most recent work along these lines [85] confirmed the occurrence of sterol-recognition sites in the TM4, TM3, and TM1 segments of each subunit, fully overlapping the lipid-exposed rings of the nAChR. The TM4 segment showed the most extensive labeling with the cholesterol analogs. ...
Pentameric ligand-gated ion channels (pLGICs) and their lipid microenvironments appear to have acquired mutually adaptive traits along evolution: 1) the three-ring architecture of their transmembrane (TM) region; 2) the ability of the outermost TM ring to convey lipid signals to the middle ring, which passes them on to the central pore ring, and 3) consensus motifs for sterol recognition in all pLGIC. Hopanoids are triterpenoid fossil lipids that constitute invaluable biomarkers for tracing evolution at the molecular scale. The cyanobacterium Gloeobacter violaceus is the oldest known living organism in which the X-ray structure of its pLGIC, GLIC, reveals the presence of the above attributes and, as discussed in this review, the ability to bind hopanoids. ELIC, the pLGIC from the bacillum Erwinia chrysanthemi is the only other known case. Both prokaryotes lack cholesterol but their pLGICs exhibit the same sterol motifs as mammalian pLGIC. This remarkable conservation suggests that the association of sterols and hopanoid surrogate molecules arose from the early need in prokaryotes to stabilize pLGIC TM regions by means of relatively rigid lipid molecules. The conservation of these phenotypic traits along such a long phylogenetic span leads us to suggest the possible co-evolution of these sterols with pLGIC.
... However, the docking studies conducted on nAChR were further validated by MD simulations, where it was found that complexes of cholesterol with nAChR were stable under these simulations [40]. These simulations were strengthened by experimental data which showed that nAChR indeed binds cholesterol at multiple sites [41]. Another example is the TRPV1 ion channel. ...
Cholesterol is essential for the proper organization of the biological membrane. Therefore, predicting which proteins can bind cholesterol is important in understanding how proteins participate in lateral membrane organization. In this study, a simple bioinformatics approach was used to establish whether MPP1, a member of the MAGUK protein family, is capable of binding cholesterol. Modelled and experimentally-validated fragment structures were mined from online resources and searched for CRAC and CRAC-like motifs. Several of these motifs were found in the primary structure of MPP1, and these were structurally visualized to see whether they localized to the protein surface. Since all of the CRAC and CRAC-like motifs were found at the surface of MPP1 domains, in silico docking experiments were performed to assess the possibility of interaction between CRAC motifs and cholesterol. The results obtained show that MPP1 can bind cholesterol via CRAC and CRAC-like motifs with moderate to high affinity (KI in the nano- to micro-molar range). It was also found that palmitoylation-mimicking mutations (C/F or C/M) did not affect the affinity of MPP1 towards cholesterol. Data presented here may help to understand at least one of the molecular mechanisms via which MPP1 affects lateral organization of the membrane.
... This approach has identified sites for a variety of non-competitive antagonists interacting with the transmembrane domain [130][131][132][133][134][135]. Affinity labelling studies have also identified binding sites for cholesterol within the nAChR transmembrane domain [136]. In addition, more indirect approaches, such as computer-docking studies, have been used. ...
Nicotinic acetylcholine receptors (nAChRs) are receptors for the neurotransmitter acetylcholine and are members of the 'Cys-loop' family of pentameric ligand-gated ion channels (LGICs). Acetylcholine binds in the receptor extracellular domain at the interface between two subunits and research has identified a large number of nAChR-selective ligands, including agonists and competitive antagonists, that bind at the same site as acetylcholine (commonly referred to as the orthosteric binding site). In addition, more recent research has identified ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for acetylcholine, including sites located in the transmembrane domain. These include positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent allosteric modulators (SAMs) and compounds that are able to activate nAChRs via an allosteric binding site (allosteric agonists). Our aim in this article is to review important aspects of the pharmacological diversity of nAChR allosteric modulators and to describe recent evidence aimed at identifying binding sites for allosteric modulators on nAChRs.
Copyright © 2015. Published by Elsevier Inc.
... However, the docking studies conducted on nAChR were further validated by MD simulations, where it was found that complexes of cholesterol with nAChR were stable under these simulations [40]. These simulations were strengthened by experimental data which showed that nAChR indeed binds cholesterol at multiple sites [41]. Another example is the TRPV1 ion channel. ...
Cholesterol is essential for the proper organization of the biological membrane. Therefore, predicting which proteins can bind cholesterol is important in understanding how proteins participate in lateral membrane organization. In this study, a simple bioinformatics approach was used to establish whether MPP1, a member of the MAGUK protein family, is capable of binding cholesterol. Modelled and experimentally-validated fragment structures were mined from online resources and searched for CRAC and CRAC-like motifs. Several of these motifs were found in the primary structure of MPP1, and these were structurally visualized to see whether they localized to the protein surface. Since all of the CRAC and CRAC-like motifs were found at the surface of MPP1 domains, in silico docking experiments were performed to assess the possibility of interaction between CRAC motifs and cholesterol. The results obtained show that MPP1 can bind cholesterol via CRAC and CRAC-like motifs with moderate to high affinity (K I in the nano-to micro-molar range). It was also found that palmitoylation-mimicking mutations (C/F or C/M) did not affect the affinity of MPP1 towards cholesterol. Data presented here may help to understand at least one of the molecular mechanisms via which MPP1 affects lateral organization of the membrane.
The muscle-type nicotinic acetylcholine receptor is a transmitter-gated ion channel residing in the plasma membrane of electrocytes and striated muscle cells. It is present predominantly at synaptic junctions, where it effects rapid depolarization of the postsynaptic membrane in response to acetylcholine released into the synaptic cleft. Previously, cryo-EM of intact membrane from Torpedo revealed that the lipid bilayer surrounding the junctional receptor has a uniquely asymmetric and ordered structure, due to a high concentration of cholesterol. It is now shown that this special lipid environment influences the transmembrane (TM) folding of the protein. All five submembrane MX helices of the membrane-intact junctional receptor align parallel to the surface of the cholesterol-ordered lipids in the inner leaflet of the bilayer; also, the TM helices in the outer leaflet are splayed apart. However in the structure obtained from the same protein after extraction and incorporation in nanodiscs, the MX helices do not align to a planar surface, and the TM helices arrange compactly in the outer leaflet. Realignment of the MX helices of the nanodisc-solved structure to a planar surface converts their adjoining TM helices into an obligatory splayed configuration, characteristic of the junctional receptor. Thus, the form of the receptor sustained by the special lipid environment of the synaptic junction is the one that mediates fast synaptic transmission; whereas, the nanodisc-embedded protein may be like the extrajunctional form, existing in a disordered lipid environment.
Membrane lipids modulate the proteins embedded in the bilayer matrix by two non-exclusive mechanisms: direct or indirect. The latter comprise those effects mediated by the physicochemical state of the membrane bilayer, whereas direct modulation entails the more specific regulatory effects transduced via recognition sites on the target membrane protein. The nicotinic acetylcholine receptor (nAChR), the paradigm member of the pentameric ligand-gated ion channel (pLGIC) superfamily of rapid neurotransmitter receptors, is modulated by both mechanisms. Reciprocally, the nAChR protein exerts influence on its surrounding interstitial lipids. Folding, conformational equilibria, ligand binding, ion permeation, topography, and diffusion of the nAChR are modulated by membrane lipids. The knowledge gained from biophysical studies of this prototypic membrane protein can be applied to other neurotransmitter receptors and most other integral membrane proteins.
Ion channels are formed by both integral transmembrane proteins and pore-forming soluble proteins. Their energetic levels in different conformational states are a function of lipid binding at hydrophobic sites in the channels, interactions between the channels and annular lipids next to their transmembrane domains, and to a lesser extent, secondary effects due to altered physical properties of annular lipids in the bilayer. Cholesterol is an abundant lipid in animal cell membranes and has been found to interact with different families of ion channels. We will review the fundamental thermodynamics behind the cholesterol-mediated gating effects on ion channels, differentiate the structural and regulatory roles of cholesterol, and use these ideas to explain the reported cholesterol-dependent effects on an array of well-studied ion channels. The principles learned here are presumably applicable to other families of membrane proteins, such as metabotropic receptors, transporters, etc.
The role of the outermost helix (M4) in the pentameric ligand-gated ion channel (pLGIC) family is currently not fully understood. It is known that M4 is important for receptor assembly, possibly via interactions with neighboring M1 and M3 helices. M4 can also transmit information on the lipid content of the membrane to the gating mechanism, and it may form a link to the extracellular domain via the Cys-loop. Our previous study examining the α4β2 nACh receptor M4 helix using HEK cells indicated M4 here is more sensitive to change than those of other pLGIC. Many of these other studies, however, were performed in Xenopus oocytes. Here we examine the nine previously identified nonfunctional α4β2 nACh receptor M4 mutant receptors using this system. The data reveal that seven of these mutant receptors do function when expressed in oocytes, with only 2, the conserved Asp at the intracellular end of M4 and a Phe in the center, having a similar phenotype (nonfunctional) in both HEK cells and oocytes. The oocyte data are more consistent with studies in other pLGIC and demonstrate the importance of the expression system used. Of the many differences between these two expression systems, we suggest that the different lipid content of the plasma membrane is a possible candidate for explaining these discrepancies.
Ion channels are formed by both integral transmembrane proteins and pore-forming soluble proteins. Their energetic levels in different conformational states are a function of lipid binding at hydrophobic sites in the channels, interactions between the channels and annular lipids next to their transmembrane domains, and to a lesser extent, secondary effects due to altered physical properties of annular lipids in the bilayer. Cholesterol is an abundant lipid in animal cell membranes and has been found to interact with different families of ion channels. We will review the fundamental thermodynamics behind the cholesterol-mediated gating effects on ion channels, differentiate the structural and regulatory roles of cholesterol and use these ideas to explain the reported cholesterol-dependent effects on an array of well-studied ion channels. The principles learned here are presumably applicable to other families of membrane proteins, such as metabotropic receptors, transporters, etc.
Nicotinic acetylcholine receptors (nAChR) are homo- or hetero-pentameric ligand-gated ion channels of the Cys-loop superfamily and play important roles in the nervous system and muscles. Studies on nAChR benefit from in silico modeling due to the lack of high-resolution structures for most receptor subtypes and challenges in experiments addressing the complex mechanism of activation involving allosteric sites. Although there is myriad of computational modeling studies on nAChR, the multitude of the methods and parameters used in these studies makes modeling nAChR a daunting task, particularly for the non-experts in the field. To address this problem, the modeling literature on Torpedo nAChR and α7 nAChR were focused on as examples of heteromeric and homomeric nAChR, and the key in silico modeling studies between the years 1995–2019 were concisely reviewed. This was followed by a critical analysis of these studies by comparing the findings with each other and with the emerging experimental and computational data on nAChR. Based on these critical analyses, suggestions were made to guide the future researchers in the field of in silico modeling of nAChR.
This article is part of the special issue on ‘Contemporary Advances in Nicotine Neuropharmacology’.
Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that the lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor’s conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.
Author Summary
Ion channels are proteins that control the flow of ions into the cell. The family of ion channels known as the pentameric ligand gated ion channels (pLGICS) open in response to the binding of a neurotransmitter, moving the channel from a resting state to an open state. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. It is also known that the response of pLGICs can also be modified by different types of lipid found within the membrane itself but exactly how is unclear. Here, we used a realistic model of a neuronal membrane and performed molecular dynamics simulations to show various lipid-protein interactions that are dependent on the channel state. Our work also reveals previously unconsidered protein-lipid interactions at a key junction of the channel known to be critical for the transmission of the opening process. We also demonstrate that cholesterol interacts with the protein at a site already known to bind to another compound that modulates the channel, called ivermectin. The work should be useful for future drug design.
Here we begin by briefly reviewing landmark structural studies on the nicotinic acetylcholine receptor. We highlight challenges that had to be overcome to push through resolution barriers, then focus on what has been gleaned in the past few years from crystallographic and single particle cryo-EM studies of different nicotinic receptor subunit assemblies and ligand complexes. We discuss insights into ligand recognition, ion permeation, and allosteric gating. We then highlight some foundational aspects of nicotinic receptor structural biology that remain unresolved and are areas ripe for future exploration.
This article is part of the special issue on ‘Contemporary Advances in Nicotine Neuropharmacology’.
Cholesterol is an integral component of cellular membranes and has been shown to be an important functional regulator for many different ion channels, including inwardly rectifying potassium (Kir) channels. Consequently, understanding the molecular mechanisms underlying this regulation represents a critical field of study. Broadly speaking, cholesterol can mediate ion channel function either directly by binding to specific sites or indirectly by altering surrounding membrane properties. Owing to the similar effects of cholesterol and its chiral isomers (epicholesterol and ent-cholesterol) on membrane properties, comparative analysis of these sterols can be an effective tool for discriminating between these direct and indirect effects. Indeed, this strategy was used to demonstrate the direct effect of cholesterol on Kir channel function. However, while this approach can discriminate between direct and indirect effects, it does not account for the promiscuity of cholesterol binding sites, which can potentially accommodate cholesterol or its chiral isomers. In this chapter, we use docking analyses to explore the idea that the specificity of cholesterol’s effect on Kir channels is dependent on the specific orientation of cholesterol within its putative binding pocket which its chiral isomers cannot replicate, even when bound themselves.
The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Cholesterol at compositions below ~33 mol percent has been shown to have negligible chemical activity, suggesting that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold. Human α1 GlyR was purified from baculovirus in-fected insect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios above and below the cholesterol ac-tivity threshold with equivalent aliquots of azi-cholesterol, a photoactivatable non-specific crosslinker. After photoactiva-tion, crosslinked cholesterol-GlyR was trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by ESI-Q-TOF MS, and sites of direct covalent attachment to peptides were refined by targeted MS/MS. Differential patterns of dozens of cholesterol-GlyR crosslinks were identified in these comparative studies, with sites of crosslinking found primarily in the fourth transmembrane helix and extramembranous connecting loops and mapping the lipid-accessible surface of the receptor. Unique crosslinking observed in both reduced and elevated cholesterol com-position suggests different apo-state structural conformations of GlyR as a function of cholesterol concentration and, in the latter studies, identified potential specific binding sites for cholesterol in the receptor.
Several essential ionotropic neurotransmitter receptors, including the nicotinic acetylcholine receptor (nAChR) and gamma-aminobutyric acid (GABA) type A receptor (GABAAr), belong to the family of pentameric ligand-gated ion channels (pLGICs). Function of these receptors is particularly sensitive to their lipid environment, including cholesterol and cholesterol-derived neurosteroids. Direct structural data investigating interactions between sterols and pLGICs, as well as their role in modulatory mechanisms, are largely unavailable. Physics-based computational approaches can serve a vital role in interpretation of more indirect data as well as hypothesis generation and experimental design. In this chapter, I report several examples in which computational approaches were used to predict direct binding interactions of steroids and pLGICs, evaluate the relative likelihood of possible interpretations of experimental data, and present rationally designed simple experiments. I conclude by offering several predictions that could be tested by future experiments.
Cholesterol is a potent modulator of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Here, we review current understanding of the mechanisms underlying cholesterol–nAChR interactions in the context of increasingly available high-resolution structural and functional data. Cholesterol and other lipids influence function by conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable vs nonactivatable conformations, as well as influencing the rates of transitions between conformational states. In the absence of cholesterol and anionic lipids, the nAChR adopts an uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions—unless the nAChR is located in a relatively thick lipid bilayer, such as that found in cholesterol-rich lipid rafts. We highlight different sites of cholesterol action, including the lipid-exposed M4 transmembrane α-helix. Cholesterol and other lipids likely alter function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These same interactions have been implicated in both the folding and trafficking of nAChRs to the cell surface. We evaluate the nature of cholesterol–nAChR interactions, considering the evidence supporting the roles of both direct binding to allosteric sites and cholesterol-induced changes in bulk membrane physical properties.
Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins
including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins
are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.
Membrane cholesterol plays an important role in modulating the function of several membrane proteins. From these proteins, a special cholesterol binding motif is reported to which the membrane cholesterol binds and modulates their activity. This consensus motif is either seen as a forward pattern known as CRAC (L/V-X(1-5)-Y-X(1-5)-R/K) and/or as a backward pattern CARC (R/K-X(1-5)-Y-X(1-5)-L/V). This as such is a low consensus motif as substituting amino acid in the unconserved positions of the motif (‘X’) yields many combinations. In order to obtain a better consensus motif for cholesterol binding, it is worthwhile to look for the same within a membrane proteins superfamily (ABC transporters, GPCRs, etc.) and assign them as a signature motif. Therefore, in the current work an attempt was made to identify the distribution of this motif in all seven helices of GPCR family and assign a consensus signature motif for an individual helix using a novel Fuzzy C-Means (FCM) approach. The workflow proceeds in four phases; first, GPCR protein sequences were extracted from UniProt database that contains seven transmembrane (TM) helices and a cholesterol dictionary has been designed for different window sizes. In second phase; those sequences are filtered which starts with R/K or L/V using both CRAC and CARC cholesterol recognition methods leading to discovery of filtered cholesterol motifs. Third phase leads to identification of significant cholesterol motifs using FCM algorithm by computing the membership of sequences to different motifs and pattern matching with different helices. Finally those uncovered cholesterol motifs that matched with TM helices were analyzed. From the results we report an algorithm that can efficiently identify and assign cholesterol signature motifs in GPCR protein sequences that can be further extended to other membrane proteins.
The concept of lipid regulation of neurotransmitter receptor function has gradually emerged in the past 40 years following pioneer studies on the nicotinic acetylcholine receptor and its interactions with cholesterol. Cholesterol, sphingolipids, and phosphoinositides interact specifically with a broad range of neurotransmitter receptors including both ionotropic and metabotropic receptors. These interactions may control the distribution of these receptors within and without plasma membrane microdomains, but they may also directly affect receptor conformation and function. In this chapter we analyze the lipid-binding properties of acetylcholine (nicotinic receptor), serotonin (5-HT1A receptor), sigma-1, NGF (Trk), and purinergic (P2X1/P2X5) receptors taken as representative examples of lipid binding and regulation of receptor function. We show that in several cases, the same receptor protein can interact with both cholesterol and sphingolipids. We focus our discussion on the molecular mechanisms associated with these interactions in the membrane environment and their impact on receptor distribution, structure, and function.
Using the crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment as a paradigm, this short overview analyzes the occurrence of structural motifs which appear not only to be conserved within the nAChR family and contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, but also extend to prokaryotic homologues found in bacteria. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TM4 ring in conveying the influence of the lipid membrane environment to the middle TM1-TM3 ring and this, in turn, to the inner TM2 channel-lining ring, which determines the ion selectivity of the channel. The preservation of these constant structural-functional features throughout such a long phylogenetic span likely points to the successful gain-of-function conferred by their early acquisition. This article is part of a Special Issue entitled: Lipid-protein interactions.
Copyright © 2015. Published by Elsevier B.V.
The plasma membrane, which encapsulates human cells, is composed of a complex mixture of lipids and embedded proteins. Emerging knowledge points towards the lipids as having a regulating role in protein function. Furthermore, insight from protein crystallography has revealed several different types of lipids intimately bound to membrane proteins and peptides, hereby possibly pointing to a site of action for the observed regulation. Cholesterol is among the lipid membrane constituents most often observed co-crystallized to membrane proteins, and the cholesterol levels in cell membranes has been found to play an essential role in health and disease. Remarkably little is known about the mechanism of lipid regulation of membrane protein function in health as well as in disease. Herein, we review molecular dynamics simulation studies aimed at investigating the effect of cholesterol on membrane protein and peptide properties. This article is part of a Special Issue entitled: Lipid-protein interactions.
Copyright © 2015. Published by Elsevier B.V.
A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.
The uncharged photoactivable probe 2-(3H)diazoflu- orene ((3H)DAF) was used to examine structural changes in the Torpedo californica nicotinic acetylcho- line receptor (AChR) ion channel induced by agonists. Photoincorporation of (3H)DAF into the AChR consisted of the following two components: a nonspecific compo- nent consistent with incorporation into residues situ- ated at the lipid-protein interface, and a specific compo- nent, inhibitable by noncompetitive antagonists and localized to the M2 hydrophobic segments of AChR sub- units. The nonspecific (3H)DAF incorporation was char- acterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled resi- dues reinforce the conclusion that the M4 segments are organized as transmembrane a-helices with a common "face" of each helix in contact with lipid. Within the M2 segments, in the absence of agonist (3H)DAF specifically labeled homologous residues bVal-261 and dVal-269, with incorporation into dVal-269 at a 5-fold greater effi- ciency than into bVal-261. This observation, coupled with the lack of detectable incorporation into a-M2 in- cluding the homologous aVal-255, indicates that within the resting channel (3H)DAF is bound with its photore- active diazo group oriented toward dVal-269. In the presence of agonist, there is an ;90% reduction in the labeling of bVal-261 and dVal-269 accompanied by spe- cific incorporation into residues (bLeu-257, bAla-258, dSer-262, and dLeu-265) situated 1 or 2 turns of an a-he- lix closer to the cytoplasmic end of the M2 segments. The results provide a further characterization of agonist- induced rearrangements of the M2 (ion channel) domain of the AChR.
We characterized the differential accessibility of the nicotinic acetylcholine receptor alpha1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or approximately 50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the beta8-beta9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128-142), the cytoplasmic loop (M3-M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane alpha-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3-M4 loop are near to the lipid bilayer.
The lipid environment of acetylcholine receptor-rich membranes from Torpedo marmorata has been studied with spin labels. The electron spin resonance spectra of both stearic acid and steroid probes in the membranes revealed an immobilized lipid component, in addition to the fluid component which is found in aqueous bilayer dispersions of the extracted lipids. The spin labels also cause a differential paramagnetic quenching of the intrinsic protein fluorescence of the membranes, which is sensitive to the action of cholinergic ligands and follows a modified Stern-Volmer law. Electron spin resonance difference spectroscopy shows that the protein-associated lipid is immobilized with respect to rotation both around and perpendicular to the long molecular axis, with correlation times : formula: (see text) approximately 50-70 ns. The proportion of lipid in the immobilized component is greater than calculated for a single boundary layer around the protein and corresponds more closely to the total interstitial lipid occupying the area between densely packed protein units in acetylcholine receptor-rich membranes.
The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.
nAChRs are pentameric transmembrane proteins into the superfamily of ligand-gated ion channels that includes the 5HT3, glycine, GABAA, and GABAC receptors. Electron microscopy, affinity labeling, and mutagenesis experiments, together with secondary structure predictions and measurements, suggest an all-beta folding of the N-terminal extracellular domain, with the connecting loops contributing to the ACh binding pocket and to the subunit interfaces that mediate the allosteric transitions between conformational states. The ion channel consists of two distinct elements symmetrically organized along the fivefold axis of the molecule: a barrel of five M2 helices, and on the cytoplasmic side five loops contributing to the selectivity filter. The allosteric transitions of the protein underlying the physiological ACh-evoked activation and desensitization possibly involve rigid body motion of the extracellular domain of each subunit, linked to a global reorganization of the transmembrane domain responsible for channel gating.
Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine
receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[3H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 μm, 3-[3H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the α-subunit in the desensitized state.
The incorporation into the α-subunit was mapped to two large proteolytic fragments. One fragment of ∼20 kDa (αV8-20), containing
the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of ∼10
kDa (αV8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within αV8-20,
the primary site of incorporation was αGlu-262 at the C-terminal end of αM2, labeled preferentially in the desensitized state.
The incorporation at αGlu-262 approached saturation between 1 μm, with ∼6% labeled, and 275 μm, with ∼30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface
at ∼1% of the levels in αGlu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional
lower affinity interactions within the agonist binding site and at the protein-lipid interface.
The free energy difference associated with the transfer of a single cholesterol molecule from the aqueous phase into a lipid bilayer depends on its final location, namely on its insertion depth and orientation within the bilayer. We calculated desolvation and lipid bilayer perturbation contributions to the water-to-membrane transfer free energy, thus allowing us to determine the most favorable location of cholesterol in the membrane and the extent of fluctuations around it. The electrostatic and nonpolar contributions to the solvation free energy were calculated using continuum solvent models. Lipid layer perturbations, resulting from both conformational restrictions of the lipid chains in the vicinity of the (rigid) cholesterol backbone and from cholesterol-induced elastic deformations, were calculated using a simple director model and elasticity theory, respectively. As expected from the amphipathic nature of cholesterol and in agreement with the available experimental data, our results show that at the energetically favorable state, cholesterol's hydrophobic core is buried within the hydrocarbon region of the bilayer. At this state, cholesterol spans approximately one leaflet of the membrane, with its OH group protruding into the polar (headgroup) region of the bilayer, thus avoiding an electrostatic desolvation penalty. We found that the transfer of cholesterol into a membrane is mainly driven by the favorable nonpolar contributions to the solvation free energy, whereas only a small opposing contribution is caused by conformational restrictions of the lipid chains. Our calculations also predict a strong tendency of the lipid layer to elastically respond to (thermally excited) vertical fluctuations of cholesterol so as to fully match the hydrophobic height of the solute. However, orientational fluctuations of cholesterol were found to be accompanied by both an elastic adjustment of the surrounding lipids and by a partial exposure of the hydrophobic cholesterol backbone to the polar (headgroup) environment. Our calculations of the molecular order parameter, which reflects the extent of orientational fluctuations of cholesterol in the membrane, are in good agreement with available experimental data.
The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitized-like state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.
To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled
nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [3H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [3H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC50 = 70 μm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [3H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential
[3H]azietomidate photoincorporation into the nAChR α and δ subunits. Photolabeled amino acids in both subunits were identified
in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized
state, there was labeling of αGlu-262 and δGln-276 at the extracellular end and δSer-258 and δSer-262 toward the cytoplasmic
end. Within the acetylcholine binding sites, [3H]azietomidate photolabeled αTyr-93, αTyr-190, and αTyr-198 in the site at the α-γ interface and δAsp-59 (but not the homologous
position, γGlu-57). Increasing [3H]azietomidate concentration from 1.8 to 150 μm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold,
consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [3H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.
A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.
The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.
A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Schägger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.
Interactions between lipids and the nicotinic acetylcholine receptor from Torpedo californica have been measured in reconstituted membranes containing purified receptor and defined lipids. The ability of brominated lipids to partially quench the intrinsic fluorescence of the acetylcholine receptor has been exploited to monitor contacts between the protein and the surrounding lipid. Relative binding constants for lipid binding to the protein have been quantitatively determined by measuring quenching observed in mixtures of brominated and nonbrominated lipids by use of equilibrium exchange equations developed by London and Feigenson [London, E., & Feigenson, G. W. (1981) Biochemistry 20, 1939-1948] and by Simmonds et al. [Simmonds, A. C., Rooney, E. K., & Lee, A. G. (1984) Biochemistry 23, 1432-1441]. Dioleoylphosphatidylcholine and its dibromo derivative are the two principal lipids used in the reconstituted membranes to establish the quenching parameters. Competition studies between cholesterol and phosphatidylcholine indicate that cholesterol does not compete effectively for the phospholipid sites presumed to surround the membrane-embedded portions of the receptor (annular lipids). However, dibromocholesterol partially quenches the receptor and leads to additional quenching of receptor in pure dibromophosphatidylcholine membranes. The results are consistent with the presence of additional binding sites for cholesterol that are not accessible to phospholipids (nonannular sites). Similar results are obtained by using cholesterol hemisuccinate and its dibromo analogue, both of which can be introduced into membranes more easily than cholesterol because of their greater solubility in water. Fatty acids appear to compete for both annular and nonannular sites, and analysis of the quenching data suggests that there are 5-10 nonannular sites associated with the receptor. Cholesterol has been shown to play a critical role in both acetylcholine receptor structural stabilization and ion channel activity, and the results presented here provide additional information about cholesterol-receptor interactions.
The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.
All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with [3H]cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Functional membranes containing purified Torpedo californica acetylcholine receptor and dioleoylphosphatidylcholine (DOPC) were prepared by a cholate dialysis procedure with lipid to protein ratios of 100-400 to 1 (mol/mol). Spin-labeled lipids were incorporated into the reconstituted membranes and into native membranes prepared from Torpedo electroplax, and electron paramagnetic resonance (EPR) spectra were recorded between 0 and 20 degrees C. The spin-labels included nitroxide derivatives of stearic acid (16-doxylstearic acid), androstane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The phospholipid spin-labels had 16-doxylstearic acid in the sn-2 position. All the spectra showed two components corresponding to a relatively mobile bilayer component and a motionally restricted "protein-perturbed" component. The relative amounts of mobile and perturbed components were quantitated by spectral subtraction and integration techniques. The mobile/perturbed ratio was somewhat temperature dependent, and the results are discussed in terms of exchange between mobile and perturbed environments. Plots of the mobile/perturbed ratios vs. lipid/protein ratios at 1 degree C gave straight lines from which the relative binding affinity of each spin-label and the number of perturbed lipids per receptor protein could be calculated. All the spin-labels gave similar values for the number of perturbed lipids (40 +/- 7), a number close to the number of lipids that will fit around the intramembranous perimeter of the receptor. The affinities of the spin-labeled lipids for the receptor relative to DOPC were androstane (K = 4.3) congruent to 16-doxylstearic acid (4.1) greater than PA (2.7) greater than PE (1.1) approximately PC (1.0) approximately PS (0.7). The lipids having the highest affinity for the acetylcholine receptor were also those that have the largest effects on the ion flux functional properties of the receptor, and the results are discussed in terms of lipid effects on receptor function.
FTIR spectra have been recorded both as a function of time and after prolonged exposure to 2H2O buffer in order to study the structural changes that lead to both the ligand- and lipid-dependent channel-inactive states of the nicotinic acetylcholine receptor (nAChR). The hydrogen/deuterium exchange spectra provide insight into both the overall rates and extent of peptide 1H/2H exchange and the individual rates and extent to which peptide hydrogens in alpha-helix and beta-sheet conformations exchange for deuterium. The spectra are also sensitive to the conformation of the polypeptide backbone and thus the secondary structure of the nAChR. The various spectral features monitored in the presence and absence of carbamylcholine and tetracaine are essentially identical, indicating that there are no large net changes in secondary structure in the channel-inactive desensitized state. The various spectral features monitored for the nAChR reconstituted into lipid membranes either with or without cholesterol are very similar, indicating that cholesterol is not a major structural regulator of the nAChR. However, in the absence of both cholesterol and anionic lipids, there is a slightly enhanced rate of exchange of alpha-helical peptide hydrogens for deuterium that occurs as a result of either an increase in nAChR dynamics or an increase in the accessibility of transmembrane peptide hydrogens to 2H2O. The latter may simply be due to an increase in the "fluidity" and thus permeability of the lipid bilayers to aqueous solvent.(ABSTRACT TRUNCATED AT 250 WORDS)
To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [125I]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [125I]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a "face" of an alpha-helix or a beta-sheet.
A photoactivatable steroid, p-azidophenacyl 3 alpha-hydroxy-5 beta-cholan-24- ate (APL), has been synthesized and used instead of cholesterol to functionally reconstitute purified acetylcholine receptor (AcChR) into vesicles made of asolectin phospholipids. Upon irradiation, the extent of AcChR photolabeling by APL is directly proportional to the amount of APL incorporated into the reconstituted vesicles and the maximum stoichiometry observed corresponds to approx. 50 mol of APL bound per mol of AcChR. Furthermore, all four subunits of the AcChR become labeled by APL and the observed labeling pattern resembles the 2:1:1:1 stoichiometry characteristic of these subunits within the AcChR complex. The presence of either cholesterol or neutral lipids from asolectin in the reconstituted bilayer decreases both, the incorporation of APl into the vesicles and the covalent labeling of the AcChR upon irradiation, without altering the stoichiometry of labeling in AcChR subunits stated above. This suggests that the potential interaction sites for the photoactivatable probe in the reconstituted AcChR are mostly those normally occupied by the natural neutral lipids. Carbamylcholine, a cholinergic agonist, also reduces the extent of APL photolabeling of the AcChR in a dose-dependent manner but, in contrast to the effects of cholesterol, the presence of carbamylcholine alters the stoichiometry of labeling in the AcChR subunits. This, along with the observation that such a decrease in the extent of APL photolabeling caused by carbamylcholine can be blocked by preincubation with alpha-bungarotoxin, suggest that AcChR desensitization induced by prolonged exposure to cholinergic agonists encompasses a rearrangement of transmembrane portions of the AcChR protein, which can be sensed by the photoactivatable probe. Conversely, presence of (+)-tubocurarine, a competitive cholinergic antagonist, has no effects on altering either the extent of APL photolabeling of the AcChR or the distribution of the labeling among AcChR subunits.
Nicotinic acetylcholine (ACh) receptors convert the binding of ACh into the opening of a cation-conducting channel. New information about the regions of the receptor most immediately involved in its function, namely the ACh-binding sites, the gate and the channel, has come from two approaches. One is the identification by labelling and by mutagenesis of residues contributing to these regions. Another is the determination of the three-dimensional structure of the receptor by electron microscopy. Although the identification of functionally relevant residues is incomplete and residues cannot yet be resolved in the three-dimensional structure, the two approaches are converging. There is still room in the gap for speculation.
The hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) has revealed important structural information about the pore of the ion channel and lipid-protein interface of the nicotinic acetylcholine receptor (AChR). To further characterize the structure of the AChR, we have mapped the sites of photoincorporation of a benzoic acid ester analogue of TID ([125I]TID-BE) and a phospholipid analogue ([125I]TIDPC/16). For each photoreactive probe, labeled sites were identified by amino-terminal sequencing of purified tryptic fragments of individual receptor subunits. [125I]TID-BE reacted with alphaCys-412, alphaMet-415, and alphaCys-418 in the M4 segment of the alpha-subunit and gammaCys-451 and gammaSer-460 in gammaM4. In the M1 segment of the alpha- and beta-subunits, [125I]TID-BE labeled alphaPhe-227, alphaLeu-228, and betaLeu-234, betaAla-235, respectively. The labeling pattern in the M1 and M4 segments indicate that TID and TID-BE interact with the AChR lipid-protein interface in a similar fashion, revealing the same lipid-exposed face of each transmembrane segment. In contrast to TID, there was, however, no detectable incorporation of [125I]TID-BE into the channel lining betaM2 segment when the AChR was labeled in the resting state conformation. In the presence of agonist (desensitized state), [125I]TID-BE reacted with betaLeu-257, betaVal-261, and beta-Leu-264 in betaM2; a labeling pattern which indicates that, in comparison to TID, the binding loci for TID-BE is located closer to the extracellular end of the channel. For [125I]TIDPC/16, receptor labeling was insensitive to the presence of agonist and the sites of incorporation mapped to the confines of the transmembrane segments alphaM4, alphaM1, and gammaM4, validating previous results found with small lipophilic probes.
A novel photoreactive analog of cholesterol, 3alpha-(4-azido-3-[125I]iodosalicylic)-cholest-5-ene ([125I]azido-cholesterol), was used to label both native acetylcholine receptor (AChR)-rich membranes from Torpedo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated [125I]azido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were initially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of [125I]azido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alphaV8-20 (alphaSer-173-Glu-338), alphaV8-10 (alphaAsn-339-Gly-439), and gammaV8-14 (gammaLeu-373-Pro-489). The digests were separated by reverse-phase high-performance liquid chromatography and labeled peptides identified by amino-terminal sequence analysis. [125I]Azido-cholesterol labeling was localized to peptides that contain almost exclusively the alpha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These results establish that the binding domain for cholesterol is at the lipid-protein interface of the AChR.
17,21-Dimethyl-19-nor-pregn-4,9-diene-3,20-dione (promegestone) was used to characterize the mechanism of inhibition of nicotinic acetylcholine (ACh) receptors (AChR) by progestin steroids. Promegestone reversibly inhibited ACh-induced currents of Torpedo AChRs expressed in Xenopus oocytes. Between 1-30 microM promegestone produced a concentration-dependent enhancement of the equilibrium binding affinity of [3H]ACh to Torpedo AChR-rich membranes. For AChRs in the presence of agonist (desensitized state) promegestone was a more potent inhibitor of the binding of the noncompetitive antagonist [3H]phencyclidine (IC50 = 9 microM) than of [3H]histrionicotoxin (IC50 approximately 100 microM). To identify AChR domains in contact with the steroid, AChR-rich membranes equilibrated with [3H]promegestone were irradiated at 312 nm, and 3H-labeled amino acids were identified by amino-terminal sequencing of fragments isolated from subunit proteolytic digests. Within AChR alpha-subunit, 70% of 3H was covalently incorporated in a 10-kDa fragment beginning at Asn-339 and containing the M4 membrane spanning segment, and 30% was in a 20-kDa fragment beginning at Ser-173 and containing the M1-M3 segments. Fragments containing the M2 channel domains as well as the M4 segments were isolated from proteolytic digests of AChR subunits and subjected to amino-terminal sequence analysis. No evidence of [3H]promegestone incorporation was detected in any of the M2 segments. The amino acids in the M4 segments labeled by [3H]promegestone were among those previously shown to be in contact with the lipid bilayer (). These results indicate that the steroid promegestone is an AChR noncompetitive antagonist that may alter AChR function by interactions at the lipid-protein interface.
To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [(3)H]diazofluorene ([(3)H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa alpha-subunit of the (Na(+)+K(+))-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by (3)H release.
6-Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6-double bond in the steroid nucleus, was used recently to identify cholesterol-binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42-49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725-1736], and to probe lipid-protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143-154]. To determine whether 6-photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.
The conversion of acetylcholine binding into ion conduction across the membrane is becoming more clearly understood in terms of the structure of the receptor and its transitions. A high-resolution structure of a protein that is homologous to the extracellular domain of the receptor has revealed the binding sites and subunit interfaces in great detail. Although the structures of the membrane and cytoplasmic domains are less well determined, the channel lining and the determinants of selectivity have been mapped. The location and structure of the gates, and the coupling between binding sites and gates, remain to be established.
The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.
The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine > tetracaine approximately procaine.
The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closed<==>open isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (phi) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. phi was constant throughout the length of the alpha subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two alpha subunits move synchronously. Between subunits, the sequence of M4 motions is alpha-epsilon-beta. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.
The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of ligand-gated ion channels (LGIC). Members of this superfamily mediate fast intercellular communication in response to endogenous neurotransmitters. This review is focused on the structural and functional crosstalk between the AChR and lipids in the membrane microenvironment, and the modulation exerted by the latter on ligand binding and ion translocation. Experimental approaches using Laurdan extrinsic fluorescence and Förster-type resonance energy transfer (FRET) that led to the characterization of the polarity and molecular dynamics of the liquid-ordered phase AChR-vicinal lipids and the bulk membrane lipids, and the asymmetry of the AChR-rich membrane are reviewed first. The topological relationship between protein and lipid moieties and the changes in physical properties induced by exogenous lipids are discussed next. This background information lays the basis for understanding the occurrence of lipid sites in the AChR transmembrane region, and the selectivity of the protein-lipid interactions. Changes in FRET efficiency induced by fatty acids, phospholipid and cholesterol (Chol), led to the identification of discrete sites for these lipids on the AChR protein, and electron-spin resonance (ESR) spectroscopy has recently facilitated determination of the stoichiometry and selectivity for the AChR of the shell lipid. The influence of lipids on AChR function is discussed next. Combined single-channel and site-directed mutagenesis data fostered the recognition of lipid-sensitive residues in the transmembrane region, dissecting their contribution to ligand binding and channel gating, opening and closing. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids.
We present a refined model of the membrane-associated Torpedo acetylcholine (ACh) receptor at 4A resolution. An improved experimental density map was obtained from 342 electron images of helical tubes, and the refined structure was derived to an R-factor of 36.7% (R(free) 37.9%) by standard crystallographic methods, after placing the densities corresponding to a single molecule into an artificial unit cell. The agreement between experimental and calculated phases along the helical layer-lines was used to monitor progress in the refinement and to give an independent measure of the accuracy. The atomic model allowed a detailed description of the whole receptor in the closed-channel form, including the ligand-binding and intracellular domains, which have not previously been interpreted at a chemical level. We confirm that the two ligand-binding alpha subunits have a different extended conformation from the three other subunits in the closed channel, and identify several interactions on both pairs of subunit interfaces, and within the alpha subunits, which may be responsible for their "distorted" structures. The ACh-coordinating amino acid side-chains of the alpha subunits are far apart in the closed channel, indicating that a localised rearrangement, involving closure of loops B and C around the bound ACh molecule, occurs upon activation. A comparison of the structure of the alpha subunit with that of AChBP having ligand present, suggests how the localised rearrangement overcomes the distortions and initiates the rotational movements associated with opening of the channel. Both vestibules of the channel are strongly electronegative, providing a cation-stabilising environment at either entrance of the membrane pore. Access to the pore on the intracellular side is further influenced by narrow lateral windows, which would be expected to screen out electrostatically ions of the wrong charge and size.
The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.
Assessing the lipid requirements of the nicotinic acetylcholine receptor
- A K Hamouda
- D Sauls
- N Vardanyan
- M Sanghvi
- M P Blanton
Hamouda AK, Sauls D, Vardanyan N, Sanghvi M, Blanton MP. Assessing the lipid requirements of
the nicotinic acetylcholine receptor. Biophys. J 2005;88:624a.