[Show abstract][Hide abstract] ABSTRACT: The mechanisms of action of endogenous modulatory ligands of connexin channels are largely unknown. Previous work showed that protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. The present study investigated the molecular mechanisms of connexin channel modulation by taurine, using hemichannels and junctional channels composed of Cx26 (homomeric) and Cx26/Cx32 (heteromeric). The addition of a 28-amino acid "tag" to the carboxyl-terminal domain (CT) of Cx26 (Cx26(T)) eliminated taurine sensitivity of homomeric and heteromeric hemichannels in cells and liposomes. Cleavage of all but four residues of the tag (Cx26(Tc)) resulted in taurine-induced pore narrowing in homomeric hemichannels, and restored taurine inhibition of heteromeric hemichannels (Cx26(Tc)/Cx32). Taurine actions on junctional channels were fully consistent with those on hemichannels. Taurine-induced inhibition of Cx26/Cx32(T) and nontagged Cx26 junctional channels was blocked by extracellular HEPES, a blocker of the taurine transporter, confirming that the taurine-sensitive site of Cx26 is cytoplasmic. Nuclear magnetic resonance of peptides corresponding to Cx26 cytoplasmic domains showed that taurine binds to the cytoplasmic loop (CL) and not the CT, and that the CT and CL directly interact. ELISA showed that taurine disrupts a pH-dependent interaction between the CT and the CT-proximal half of the CL. These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function.
The Journal of General Physiology 08/2011; 138(3):321-39. DOI:10.1085/jgp.201110634 · 4.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.
Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.
This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.
[Show abstract][Hide abstract] ABSTRACT: Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; MS) and MALDI-TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3% by MS, with 29.9% by MS/MS. MS coverage was 92.6% if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, gamma-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26-Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.
[Show abstract][Hide abstract] ABSTRACT: Intercellular channels formed by connexin proteins play a pivotal role in the direct movement of ions and larger cytoplasmic solutes between vascular endothelial cells, between vascular smooth muscle cells, and between endothelial and smooth muscle cells. Multiple genetic and epigenetic factors modulate connexin expression levels and/or channel function, including cell-type-independent and cell-type-specific transcription factors, posttranslational modifications, and localized membrane targeting. Additionally, differences in protein-protein interactions, including those between connexins, significantly contribute to both vascular homeostasis and disease progression. The biophysical properties of the connexin channels identified in the vasculature, those formed by Cx37, Cx40, Cx43 and/or Cx45 proteins, are discussed in this chapter in the physiological and pathophysiological context of vessel function.
International review of cell and molecular biology 01/2009; 278:69-118. DOI:10.1016/S1937-6448(09)78002-5 · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because of the diversity of connexin isoforms and their combinations that can compose connexin channels, there is large diversity
in the permeability properties of the channels. Unlike most ion channels, the relevant permeabilities extend from charge selectivity
among atomic ions such as K+ and Ca2+, through the size and charge selectivity among nonbiological tracer molecules, to highly specific selectivities among cytoplasmic
molecules. Distinct experimental approaches are used to define each of these types of selectivity. In general, permeability
to current-carrying atomic ions is high, but is substantially charge-selective in some cases. In general, there is permeability
to molecular tracers up to ~800Da, with some channels having significantly lower cutoff thresholds, influenced by charge.
Permeability to cytoplasmic molecules is less well explored, but seems to be highly variable and specific, often violating
the size and charge selectivities suggested by studies using nonbiological tracers. The unitary channel conductances and the
molecular permeabilities to tracer molecules and to cytoplasmic molecules do not correlate well with each other, and do not
allow easy inferences or extrapolations about what biological molecules can permeate any particular form of connexin channel,
and how well. In spite of this, even the sparse data obtained to date on cytoplasmic permeants gives clues as to the roles
that connexin-specific permeabilities may play in biology.
KeywordsConnexin-Gap junction-Unitary conductance-Pore width-Charge selectivity-Tracer permeability-Molecular permeability-Second messengers
[Show abstract][Hide abstract] ABSTRACT: Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse.
Cell and Tissue Research 05/2007; 328(1):97-107. DOI:10.1007/s00441-006-0301-6 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The isoelectric points of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) were determined by isoelectric focusing in free fluids. The isoelectric points were significantly more acidic than predicted from amino acid sequences and different from each other, allowing homomeric channels to be resolved separately. The isoelectric points of the homomeric channels bracketed the isoelectric points of heteromeric Cx26/Cx32 channels. For heteromeric channels, Cx26 and Cx32 were found in overlapping, pH-focused fractions, indicating quaternary structure was retained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify post-translational modifications of Cx26 and Cx32 cytoplasmic domains, including the first reported post-translational modifications of Cx26. Suspected modifications were hydroxylation and/or phosphorylation near the amino terminus of both connexins, gamma-carboxyglutamate residues in the cytoplasmic loop of both connexins, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation at the carboxyl-terminus of Cx32. These modifications contribute to the measured acidic isoelectric points of Cx26 and Cx32, whereas their low molecular masses would not appreciably change connexin SDS-PAGE mobility. Most of these modifications have not previously been identified for connexins and may be instrumental in guiding and understanding novel aspects of channel trafficking and molecular mechanisms of channel regulation.
The FASEB Journal 07/2006; 20(8):1221-3. DOI:10.1096/fj.05-5309fje · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous work has shown that channels formed by both connexin (Cx)26 and Cx32 (heteromeric Cx26/Cx32 hemichannels) are selectively permeable to cAMP and cGMP. To further investigate differential connexin channel permeability among second messengers, and the influence of connexin channel composition on the selectivity, the permeability of inositol phosphates with one to four phosphate groups through homomeric Cx26, homomeric Cx32, and heteromeric Cx26/Cx32 channels was examined. Connexin channels were purified from transfected HeLa cells and from rat, mouse, and guinea pig livers, resulting in channels with a broad range of Cx26/Cx32 aggregate ratios. Permeability to inositol phosphates was assessed by flux through reconstituted channels. Surprisingly, myoinositol and all inositol phosphates tested were permeable through homomeric Cx32 and homomeric Cx26 channels. Even more surprising, heteromeric Cx26/Cx32 channels showed striking differences in permeability among inositol phosphates with three or four phosphate groups and among isomers of inositol triphosphate. Thus, heteromeric channels are selectively permeable among inositol phosphates, whereas the corresponding homomeric channels are not. There was no discernible difference in the permeability of channels with similar Cx26/Cx32 ratios purified from native and heterologous sources. The molecular selectivity of heteromeric channels among three inositol triphosphates could not be accounted for by simple connexin isoform stoichiometry distributions and therefore may depend on specific isoform radial arrangements within the hexameric channels. Dynamic regulation of channel composition in vivo may effectively and efficiently modulate intercellular signaling by inositol phosphates.
[Show abstract][Hide abstract] ABSTRACT: Cell extraction with cold nonionic detergents or alkaline carbonate prepares an insoluble membrane fraction whose buoyant density permits its flotation in discontinuous sucrose gradients. These lipid "rafts" are implicated in protein sorting and are attractive candidates as platforms that coordinate signal transduction pathways with intracellular substrates. Gap junctions form a direct molecular signaling pathway by end-to-end apposition of hemichannels containing one (homomeric) or more (heteromeric) connexin isoforms. Residency of channels composed of Cx26 and/or Cx32 in lipid rafts was assessed by membrane insolubility in alkaline carbonate or different concentrations of Triton X100, Nonidet P40 and Brij-58 nonionic detergents. Using Triton X100, insoluble raft membranes contained homomeric Cx32 channels, but Cx26-containing channels only when low detergent concentrations were used. Results were similar using Nonidet P40, except that Cx26-containing channels were excluded from raft membranes at all detergent concentrations. In contrast, homomeric Cx26 channels were enriched within Brij-58-insoluble rafts, whereas Cx32-containing channels partitioned between raft and nonraft membranes. Immunofluorescence microscopy showed prominent colocalization only of nonjunctional connexin channels with raft plasma membrane; junctional plaques were not lipid rafts. Rafts prepared by different extraction methods had considerable quantitative and qualitative differences in their lipid compositions. That functionally different nonjunctional connexin channels partition among rafts with distinct lipid compositions suggests that unpaired Cx26 and/or Cx32 channels exist in membrane domains of slightly different physicochemical character. Rafts may be involved in trafficking of plasma membrane connexin channels to gap junctions.
[Show abstract][Hide abstract] ABSTRACT: Abrupt developmental changes occur in structural form and function of connexin (Cx) channels in the mouse mammary gland. Microarray study shows that the principal connexin isoform in epithelial cells during pregnancy is Cx26, up-regulated and persisting from the virgin. After parturition, there is rapid induction of Cx32. In epithelial plasma membranes, size exclusion chromatography reveals that Cx32 organizes initially with Cx26 as heteromeric (Cx26-Cx32) hemichannels and later in heteromeric and homomeric Cx32 channels. Dramatic alterations of connexin channel function following these developmental changes in channel composition are characterized using native channels reconstituted into liposomes. Changes to channel stoichiometry increase the allowable physical size limits of permeant after parturition; the new Cx32 channels are wider than channels containing Cx26. Most remarkably, heteromeric Cx26-Cx32 channels are selectively permeability to adenosine 3',5' cyclic phosphate (cAMP), guanosine 3',5' cyclic phosphate (cGMP), and inositol 1,4,5-triphosphate (IP(3)), whereas homomeric channels are not. Homomeric Cx26 and heteromeric channels with high Cx26/Cx32 stoichiometry are also inhibited by taurine, an osmolyte playing a key role in milk protein synthesis. Taurine effect is reduced where heteromeric channels contain Cx32 > Cx26 and eliminated when channels contain only Cx32. Connexin channel stoichiometry, permeability, and chemical gating character change in precisely the desired fashion after parturition to maximize molecular and electrical coupling to support coordinated milk secretion.
Experimental Cell Research 09/2004; 298(2):643-60. DOI:10.1016/j.yexcr.2004.05.003 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclodextrins (CDs), a series of hollow cyclic glucosaccharides, can reversibly block molecular permeation through channels formed by connexin-32 and/or connexin-26 reconstituted into liposomes. The character of the block changes as a function of the size of the CD relative to the connexin pore diameter, suggesting that the block occurs via entry of the CD into the pore lumen and occlusion of the permeability pathway. The block occurs only when the CD is applied to the side of the pore that is normally cytoplasmic and not from the side that is normally extracellular. The block is potentiated when organic analytes are sequestered in the hydrophobic interior of the CDs. CDs may be useful as molecular tools with which to explore the structure of the connexin pore and to alter molecular movement through connexin channels.
[Show abstract][Hide abstract] ABSTRACT: To facilitate the use of oligosaccharides as analytical tools in biological studies, we have designed, synthesized, and conjugated to maltosaccharides a novel series of homologous small fluorescent moieties that differ in formal charge. These moieties are amide derivatives of anthranilic acid: uncharged N-(2-aminobenzoyl)glycinamide (ABGlyAmide; 2), acidic N,N-dimethyl-N(')-(2-aminobenzoyl)ethylenediamine (ABGlyDIMED; 3), and basic N-(2-aminobenzoyl)glycine (ABGly; 1). Routes for synthesis and optimal reaction conditions for glycoconjugation by conventional reductive amination are presented, as is the compatibility of these adducts with common analytical and preparative chromatographic methods, including RP-HPLC and HPAEC-PAD. These novel anthranilic acid derivatives confer both fluorescence and defined charge to oligosaccharides, and so enhance the repertoire of chromatographic and analytical methods for which anthranilic acid can be used. Furthermore, because glucosaccharides have rigid solution structure, these small fluorescent adducts with different formal charge are ideal tools for molecular sizing studies of membrane pores.
Carbohydrate Research 02/2004; 339(2):221-31. DOI:10.1016/j.carres.2003.10.020 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.
[Show abstract][Hide abstract] ABSTRACT: Progress in the characterization of gap junctions and their constituent connexin sub-units is leading to a greater understanding of the structure, function, and regulation of this cell-cell communication channel. Although much of the experimental evidence generated to date is correlative, recent work utilizing reverse genetic approaches to manipulate connexin gene function has provided direct evidence that intercellular communication via gap junctions plays key roles in development, cellular differentiation, and organogenesis. Pathogenic mutations in human connexin genes have now been identified. Furthermore, a considerable body of experimental evidence correlates a loss of junctional communication with progression to a malignant phenotype. Although the cell biology of the mammary gland has been extensively studied, the role(s) of gap junctions in the development, differentiation, and maintenance of this tissue are unknown. Gap junctions were first reported in the mammary gland following freeze-fracture and electron microscopic analyses. The development of anti-connexin antibodies and the cloning of individual connexin isoforms have enabled this work to be extended, but there are contradictory reports in the temporal expression patterns of these proteins within mammary epithelium. In addition, a recent report in this Journal has implied by immunocytochemistry that there is up-regulation of connexin protein in some human breast tumours, a novel observation which may be inconsistent with the proposed tumour suppressor role for gap junctions.
The Journal of Pathology 12/1998; 186(4):343-9. DOI:10.1002/(SICI)1096-9896(199812)186:4<343::AID-PATH189>3.0.CO;2-X · 7.43 Impact Factor