[Show abstract][Hide abstract] ABSTRACT: Proteoglycan spatiotemporal organization underpins extracellular matrix biology but atomic scale glimpses of this microarchitecture are obscured by glycosaminoglycan size and complexity. To overcome this, multi-microsecond aqueous simulations of chondroitin and dermatan sulfates were abstracted into a prior coarse-grained model, which was extended to heterogeneous glycosaminoglycans and small leucine-rich proteoglycans. Exploration of relationships between sequence and shape led to hypotheses that proteoglycan size is dependent on glycosaminoglycan unit composition but independent of sequence permutation. Uronic acid conformational equilibria were modulated by adjacent hexosamine sulfonation and iduronic acid increased glycosaminoglycan chain volume and rigidity, while glucuronic acid imparted chain plasticity. Consequently, block copolymeric glycosaminoglycans contained microarchitectures capable of multivalent binding to growth factors and collagen, with potential for interactional synergy at greater chain number. The described atomic scale views of proteoglycans and heterogeneous glycosaminoglycans provide structural routes to understanding their fundamental signaling and mechanical biological roles and development of new biomaterials.
[Show abstract][Hide abstract] ABSTRACT: Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.
Preview · Article · Mar 2014 · Physical Chemistry Chemical Physics
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes
to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6
consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this
high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based
on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex
using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides
(from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that
a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes
the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism
for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated
transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.
Full-text · Article · Jan 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The human glycome comprises a vast untapped repository of 3D-structural information that holds the key to glycan recognition and a new era of rationally designed mimetic chemical probes, drugs, and biomaterials. Toward routine prediction of oligosaccharide conformational populations and exchange rates at thermodynamic equilibrium, we apply hardware-accelerated aqueous molecular dynamics to model μs motions in N-glycans that underpin inflammation and immunity. In 10μs simulations, conformational equilibria of mannosyl cores, sialyl Lewis (sLe) antennae, and constituent sub-sequences agreed with prior refinements (X-ray and NMR). Glycosidic linkage and pyranose ring flexing were affected by branching, linkage position, and secondary structure, implicating sequence dependent motions in glycomic functional diversity. Linkage and ring conformational transitions that have eluded precise quantification by experiment and conventional (ns) simulations were predicted to occur on μs timescales. All rings populated non-chair shapes and the stacked galactose and fucose pyranoses of sLe(a) and sLe(x) were rigidified, suggesting an exploitable 3D-signature of cell adhesion protein binding. Analyses of sLe(x) dynamics over 25μs revealed that only 10μs were sufficient to explore all aqueous conformers. This simulation protocol, which yields conformational ensembles that are independent of initial 3D-structure, is proposed as a route to understanding oligosaccharide recognition and structure-activity relationships, toward development of carbohydrate-based novel chemical entities.
Preview · Article · Oct 2013 · Carbohydrate research
[Show abstract][Hide abstract] ABSTRACT: The biological information encoded in carbohydrate sequences dwarfs that of proteins and nucleic acids. Deciphering structure-function relationships in heparin and heparan sulfate (the heparanome) is further compounded by extreme sequence diversity, experimental difficulties and the computational cost of rigorous modeling. Here, we perform unbiased microsecond dynamics simulations of 11 heparanome oligosaccharides (55 microseconds total) to investigate the effect of sequence on 3D-structure and to underpin a coarse-grained model that is consistent with long-timescale experimentally-validated atomic motions in water. Pyranose ring flexing (puckering) in 2-O-sulfo-α-L-iduronic acid, which underlies heparin-mediated anticoagulation, was modulated by polymerization (chain position and adjacent residues), which is supported by previous experiments. Furthermore, in coarse-grained simulations inclusion of puckering was essential to predict macroscopic hydrodynamic properties of heparan sulfate chains containing hundreds of monosaccharaides. Our structural findings and model enable rational molecular design and we propose that in the heparanome, puckering, polymer 3D-shape and bioactivity are inextricably linked.
No preview · Article · Feb 2013 · Biomacromolecules
[Show abstract][Hide abstract] ABSTRACT: In the aldohexopyranose idose, the unique presence of three axial ring hydroxyl groups causes considerable conformational flexibility, rendering it challenging to study experimentally and an excellent model for rationalizing the relationship between puckering and anomeric configuration. Puckering in methyl α- and β-L-idopyranosides was predicted from kinetically rigorous 10 μs simulations using GLYCAM11 and three explicit water models (TIP3P, TIP4P, and TIP4P-EW). In each case, computed pyranose ring three-bond (vicinal) (1)H-(1)H spin couplings ((3)J(H,H)) trended with NMR measurements. These values, calculated puckering exchange rates and free energies, were independent of the water model. The α- and β-anomers were (1)C(4) chairs for 85 and >99% of their respective trajectories and underwent (1)C(4)→(4)C(1) exchange at rates of 20 μs(-1) and 1 μs(-1). Computed α-anomer (1)C(4)↔(4)C(1) puckering rates depended on the exocyclic C6 substituent, comparing hydroxymethyl with carboxyl from previous work. The slower kinetics and restricted pseudorotational profile of the β-anomer were caused by water occupying a cavity bounded by the anomeric 1-O-methyl and the C6 hydroxymethyl groups. This finding rationalizes the different methyl α- and β-L-idopyranoside (3)J(H,H) values. Identifying a relationship between idopyranose anomeric configuration, microsecond puckering, and water structure facilitates engineering of biologically and commercially important derivatives and underpins deciphering presently elusive structure-function relationships in the glycome.
Preview · Article · May 2012 · The Journal of Physical Chemistry B
[Show abstract][Hide abstract] ABSTRACT: Reconciling glycocodes and their associated bioactivities, via 3D-structure, will rationalise burgeoning high-throughput functional glycomics data and underpin a new era of opportunity in chemical biology. A major impasse to achieving this goal is a detailed understanding of pyranose sugar ring 3D-conformation (or pucker) and the affiliated microsecond-timescale exchange kinetics. Here, we perform hardware-accelerated kinetically-rigorous equilibrium simulations of fundamental monosaccharides to produce the hypothesis that pyranoses have microsecond-timescale kinetic puckering signatures in water, classified as unstable (rare in the glycome), metastable (infrequently observed) and stable (prevalent). The predicted μs-metastability of β-d-glucose explained hitherto irreconcilable experimental measurements. Twisted puckers seen in carbohydrate enzymes were present in the aqueous 3D-ensemble (suggesting preorganization) and pyranose-water interactions accounted for the relative stability of β-d-galactose. Characteristic 3D-shapes for biologically- and commercially-important carbohydrates and new rules linking chemical modifications with pyranose μs-puckering kinetics are proposed. The observations advance structural-glycomics towards dynamic 3D-templates suitable for structure-based design.
No preview · Article · Mar 2012 · Physical Chemistry Chemical Physics
[Show abstract][Hide abstract] ABSTRACT: Understanding microsecond-timescale dynamics is crucial to establish three-dimensional (3D) structure–activity relationships
in sugars but has been intractable to experiments and simulations. As a consequence, whether arguably the most important chemical
scaffold in glycobiology, N-acetyl-d-glucosamine (GlcNAc), deviates from a rigid 4C1 chair is unknown. Here, conformer populations and exchange kinetics were quantified from the longest aqueous carbohydrate
simulations to date (0.2 ms total) of GlcNAc, four derivatives from heparan sulfate and their methylglycosides. Unmodified
GlcNAc took 3–5 μs to reach a conformational equilibrium, which comprised a metastable 4C1 chair that underwent 4C1 ↔ 1C4 transitions at a predicted forward rate of 0.8 μs−1 with an average 1C4-chair lifetime of 3 ns. These predictions agree with high-resolution crystallography and nuclear magnetic resonance but not
with the hypothesis that GlcNAc is a rigid 4C1 chair, concluded from previous experimental analyses and non-aqueous modeling. The methylglycoside was calculated to have
a slower forward rate (0.3 μs−1) and a more stable 4C1 conformer (0.2 kcal mol−1), suggesting that pivotal 3D intermediates (particularly 2SO, 1S5 and B2,5) increased in energy, and water was implicated as a major cause. Sulfonation (N-, 3-O and 6-O) significantly augmented this effect by blocking pseudorotation, but did not alter the rotational preferences of hydroyxl
or hydroxymethyl groups. We therefore propose that GlcNAc undergoes puckering exchange that is dependent on polymerization
and sulfo substituents. Our analyses, and 3D model of the equilibrium GlcNAc conformer in water, can be used as dictionary
data and present new opportunities to rationally modify puckering and carbohydrate bioactivity, with diverse applications
from improving crop yields to disease amelioration.
[Show abstract][Hide abstract] ABSTRACT: The 3D-structure of extracellular matrix glycosaminoglycans is central to function, but is currently poorly understood. Resolving this will provide insight into their heterogeneous biological roles and help to realize their significant therapeutic potential. Glycosaminoglycan chemical isoforms are too numerous to study experimentally and simulation provides a tractable alternative. However, best practice for accurate calculation of glycosaminoglycan 3D-structure within biologically relevant nanosecond timescales is uncertain. Here, we evaluate the ability of three potentials to reproduce experimentally observed glycosaminoglycan monosaccharide puckering, disaccharide 3D-conformation, and characteristic solvent interactions. Temporal dynamics of unsulfated chondroitin, chondroitin-4-sulfate, and hyaluronan β(1→3) disaccharides were simulated within TIP3P explicit solvent unrestrained for 20 ns using the GLYCAM06 force-field and two semi-empirical quantum mechanics methods, PM3-CARB1 and SCC-DFTB-D (both within a hybrid QM/MM formalism). Comparison of calculated and experimental properties (vicinal couplings, nuclear Overhauser enhancements, and glycosidic linkage geometries) showed that the carbohydrate-specific parameterization of PM3-CARB1 imparted quantifiable benefits on monosaccharide puckering and that the SCC-DFTB-D method (including an empirical correction for dispersion) best modeled the effects of hexosamine 4-sulfation. However, paradoxically, the most approximate approach (GLYCAM06/TIP3P) was the best at predicting monosaccharide puckering, 3D-conformation, and solvent interactions. Our data contribute to the debate and emerging consensus on the relative performance of these levels of theory for biological molecules.
No preview · Article · Dec 2010 · Journal of Computational Chemistry
[Show abstract][Hide abstract] ABSTRACT: The pyranose ring of L-iduronic acid (IdoA), a major constituent of the anticoagulant heparin, is an equilibrium of multiple ring puckers that have evaded quantification by experiment or computation. In order to resolve this enigma, we have calculated the free energy landscape of IdoA and two related monosaccharides from extensive microsecond simulations. After establishing that the simulated puckers had reached equilibrium, hypotheses were confirmed that (a) IdoA (1)C(4)- and (4)C(1)-chair conformations exchange on the microsecond time scale, (b) C5 epimerization leads to a (4)C(1)-chair, and (c) IdoA 2-O-sulfation (IdoA2S) stabilizes the (1)C(4) conformer. The IdoA and IdoA2S (1)C(4) conformers were isoenergetic and computed to be 0.9 and 2.6 kcal mol(-1) lower in free energy than their respective (4)C(1)-chair conformations. The simulations also predicted that the IdoA (2)S(O)-skew-boat was less populated than previously thought. Novel chemical synthesis and ultra-high-field NMR supported these observations, but slight discrepancies in observed and predicted NMR vicinal couplings implied that the simulation overestimated the population of the IdoA (4)C(1)-chair with respect to (1)C(4)-chair due to small force field inaccuracies that only manifest in long simulations. These free-energy calculations drive improvements in computational methods and provide a novel route to carbohydrate mimetic biomaterials and pharmaceuticals.
No preview · Article · Sep 2010 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-structure at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without (15)N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the beta(1-->3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the beta(1-->3) psi dihedral (as previously predicted by simulation), while the beta(1-->4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.
Preview · Article · Nov 2009 · Carbohydrate research
[Show abstract][Hide abstract] ABSTRACT: The human alpha7 nicotinic acetylcholine receptor (nAChR) subunit and its Caenorhabditis elegans homolog, ACR-16, can generate functional recombinant homomeric receptors when expressed in Xenopus laevis oocytes. Both nAChRs express robustly in the presence of the co-injected chaperone, RIC-3, and show striking differences in the actions of a type I positive allosteric modulator (PAM), ivermectin (IVM). Type I PAMs are characterised by an increase in amplitude only of the response to acetylcholine (ACh), whereas type II PAMs exhibit, in addition, changes in time-course/desensitization of the ACh response. The type I PAMs, ivermectin, 5-hydroxyindole (5-HI), NS-1738 and genistein and the type II PAM, PNU-120596, are all active on human alpha7 but are without PAM activity on ACR-16, where they attenuate the amplitude of the ACh response. We used the published structure of avermectin B1a to generate a model of IVM, which was then docked into the candidate transmembrane allosteric binding site on alpha7 and ACR-16 in an attempt to gain insights into the observed differences in IVM actions. The new pharmacological findings and computational approaches being developed may inform the design of novel PAM drugs targeting major neurological disorders.
[Show abstract][Hide abstract] ABSTRACT: The molecular diversity of many gene products functioning in the nervous system is enhanced by alternative splicing and adenosine-to-inosine editing of pre-mRNA. Using RDL, a Drosophila melanogaster GABA-gated ion channel, we examined the functional impact of RNA editing at several sites along with alternative splicing of more than one exon. We show that alternative splicing and RNA editing have a combined influence on the potency of the neurotransmitter GABA, and the editing isoforms detected in vivo span the entire functional range of potencies seen for all possible edit variants expressed in Xenopus laevis oocytes. The extent of RNA editing is developmentally regulated and can also be linked to the choice of alternative exons. These results provide insights into how the rich diversity of signaling necessary for complex brain function can be achieved by relatively few genes.
Full-text · Article · May 2009 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The tricyclic isoalloxazine nucleus of the redox cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) acts as an electron sink in life-sustaining biological electron transfer (eT). The functional diversity of flavin-containing proteins (flavoproteins) transcends that of free flavins. A large body of experimental evidence attributes natural control of flavoprotein-mediated eT to tuning of the thermodynamic driving force by the protein environment. Understanding and engineering such modulation by the protein environment of the flavin redox potential (DeltaE(o)) is valuable in biotechnology and device design. In this study we employed classical molecular dynamics free energy simulations (MDFES), within a thermodynamic integration (TI) formalism, to calculate the change in FMN first reduction potential (DeltaDeltaE(o)(ox/sq)) imparted by 6 flavoprotein active site mutations. The combined performance of the AMBER ff03 (protein) and GAFF (cofactor) force fields was benchmarked against experimental data for mutations close to the isoalloxazine re- and si-faces that perturb the wild-type DeltaE(o)(ox/sq) value in Anabaena flavodoxin. The classical alchemical approach used in this study overestimates the magnitude of DeltaE(o) values, in common with other studies. Nevertheless, chemically accurate DeltaDeltaE(o) values--calculated to within 1 kcal mol(-1) of the experimental value--were obtained for five of the six mutations studied. We have shown that this approach is practical for quantitative in silico screening of the effect of mutations on the first reduction potential where experimental values and structural data are available for the wild-type flavoprotein. This approach promises to be useful as an integral part of future interdisciplinary strategies to engineer desired thermodynamic properties in flavoproteins of biotechnological interest.
No preview · Article · Nov 2008 · The Journal of Physical Chemistry A
[Show abstract][Hide abstract] ABSTRACT: Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p'-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); gamma-aminobutyric acid (GABA) receptors (cyclodienes, gamma-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.
No preview · Article · Dec 2005 · Invertebrate Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Ionotropic GABA receptors are abundant in both vertebrate and invertebrate nervous systems, where they mediate rapid, mostly inhibitory synaptic transmission. A GABA-gated chloride channel subunit from Drosophila melanogaster [Resistant to Dieldrin (RDL)] has been cloned, functionally expressed, and found to exhibit many aspects of the pharmacology of native, bicuculline-insensitive insect GABA receptors. RDL is the target of the commercially important insecticide fipronil. A point mutation in the channel-lining region of the RDL molecule is known to underlie most cases of resistance to insecticides acting on GABA receptors. RDL is widely distributed throughout the insect nervous system, but the subunit composition of RDL-containing in native receptors is unknown. It is possible that in some instances, RDL coexpresses with glutamate-gated chloride channel subunits. Other ionotropic receptor subunits (LCCH3 and GRD) form GABA-gated cation channels when heterologously expressed. Interest in RDL as a model ligandgated anion channel has been enhanced by the recent discovery of pre-mRNA A-to-I editing, which, together with alternative splicing, adds to the functional diversity of this GABA receptor subunit.
Preview · Article · Nov 2005 · Molecular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The reactions of several active site mutant forms of bacterial morphinone reductase (MR) with NADH and 2-cyclohexen-1-one as substrates have been studied by stopped-flow and steady-state kinetic methods and redox potentiometry. The enzymes were designed to (i) probe a role for potential proton donors (Tyr-72 and Tyr-356) in the oxidative half-reaction of MR; (ii) assess the function of a highly conserved tryptophan residue (Trp-106) in catalysis; (iii) investigate the role of Thr-32 in modulating the FMN reduction potential and catalysis. The Y72F and Y356F enzymes retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key proton donors in the oxidative reaction of MR. Taken together with our recently published data (Messiha, H. L., Munro, A. W., Bruce, N. C., Barsukov, I., and Scrutton, N. S. (2005) J. Biol. Chem. 280, 4627-4631) that rule out roles for Cys-191 (corresponding with the proton donor, Tyr-196, in the structurally related OYE1 enzyme) and His-186 as proton donors, we infer solvent is the source of the proton in the oxidative half-reaction of MR. We demonstrate a key role for Thr-32 in modulating the reduction potential of the FMN, which is decreased approximately 50 mV in the T32A mutant MR. This effects a change in rate-limiting step in the catalytic cycle of the T32A enzyme with the oxidizing substrate 2-cyclohexenone. Despite the conservation of Trp-106 throughout the OYE family, we show this residue does not play a major role in catalysis, although affects on substrate and coenzyme binding are observed in a W106F enzyme. Our studies show some similarities, but also major differences, in the catalytic mechanism of MR and OYE1, and emphasize the need for caution in inferring mechanism by structural comparison of highly related enzymes in the absence of solution studies.
Full-text · Article · Aug 2005 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are important for fast synaptic cholinergic transmission. They are targets of drugs/chemicals for human and animal health as well as for pest control. With the advent of genome sequencing, entire nAChR gene families have now been described for vertebrates and invertebrates. Mostly, these are extensive with a large number of distinct subunits, making possible many nAChR subtypes differing in transmitter affinity, channel conductance, ion selectivity, desensitization, modulation and pharmacology. The smallest nAChR gene family to date is that of the fruit fly, Drosophila melanogaster, with only 10 members. This apparently compact family belies its true diversity as 4 of the 10 subunits show alternative splicing. Also, using Drosophila, A-to-I pre-mRNA editing has been demonstrated for the first time in nAChRs. Such is the extent of this variation, that one subunit alone (Dalpha6) can potentially generate far more isoforms than seen in entire gene families from other species. We present here three-dimensional models constructed for insect nAChRs, which show that many variations introduced by alternative splicing and RNA editing may influence receptor function.
[Show abstract][Hide abstract] ABSTRACT: RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wild-type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor by GABAergic insecticides.
No preview · Article · Feb 1996 · Neuropharmacology