Postsynaptic receptors regulate presynaptic transmitter stability through transsynaptic bridges.

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABAA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABAA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation.

Pentameric ligand-gated ion channels (pLGICs) are the targets of several clinical and endogenous allosteric modulators including anesthetics and neurosteroids. Molecular mechanisms underlying allosteric drug modulation are poorly understood. Here, we constructed a chimeric pLGIC by fusing the extracellular domain (ECD) of the proton-activated, cation-selective bacterial channel GLIC to the transmembrane domain (TMD) of the human ρ1 chloride-selective GABAAR, and tested the hypothesis that drug actions are regulated locally in the domain that houses its binding site. The chimeric channels were proton-gated and chloride-selective demonstrating the GLIC ECD was functionally coupled to the GABAρ TMD. Channels were blocked by picrotoxin and inhibited by pentobarbital, etomidate and propofol. The point mutation, ρ TMD W328M, conferred positive modulation and direct gating by pentobarbital. The data suggest that the structural machinery mediating general anesthetic modulation resides in the TMD. Proton-activation and neurosteroid modulation of the GLIC-ρ chimeric channels, however, did not simply mimic their respective actions on GLIC and GABAρ revealing that across domain interactions between the ECD and TMD play important roles in determining their actions. Proton-induced current responses were biphasic suggesting that the chimeric channels contain an additional proton sensor. Neurosteroid modulation of the GLIC-ρ chimeric channels by the stereoisomers, 5α-THDOC and 5ß-THDOC, were swapped compared to their actions on GABAρ indicating that positive versus negative neurosteroid modulation is not encoded solely in the TMD nor by neurosteroid isomer structure but is dependent on specific interdomain connections between the ECD and TMD. Our data reveal a new mechanism for shaping neurosteroid modulation.

The endoplasmic reticulum-based acetyltransferases, ATase1 and ATase2, associate with the oligosaccharyltransferase to acetylate correctly folded polypeptides.

The endoplasmic reticulum (ER) has two membrane-bound acetyltransferases responsible for the endoluminal N(ϵ)-lysine acetylation of ER-transiting and -resident proteins. Mutations that impair the ER-based acetylation machinery are associated with developmental defects and a familial form of spastic paraplegia. Deficient ER acetylation in the mouse leads to defects of the immune and nervous system. Here, we report that both ATase1 and ATase2 form homo- and heterodimers and associate with members of the oligosaccharyltransferase (OST) complex. In contrast to the OST, the ATases only modify correctly folded polypetides. Collectively, our studies suggest that one of the functions of the ATases is to work in concert with the OST and "select" correctly folded from unfolded/misfolded transiting polypeptides.

Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions.

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Šinward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2-M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating.

Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1ß2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying pLGIC gating transitions.

Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Structural link between γ-aminobutyric acid type A (GABAA) receptor agonist binding site and inner ß-sheet governs channel activation and allosteric drug modulation.

Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner ß-sheet trigger channel activation. Homology modeling reveals that the ß4-ß5 linker physically connects the neurotransmitter binding site to the inner ß-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor ß-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.

Different residues in the GABAA receptor benzodiazepine binding pocket mediate benzodiazepine efficacy and binding.

Benzodiazepines (BZDs) exert their therapeutic actions by binding to the GABA(A) receptor (GABA(A)R) and allosterically modulating GABA-induced chloride currents (I(GABA)). A variety of ligands with divergent structures bind to the BZD site, and the structural mechanisms that couple their binding to potentiation of I(GABA) are not well understood. In this study, we measured the effects of individually mutating 22 residues throughout the BZD binding pocket on the abilities of eszopiclone, zolpidem, and flurazepam to potentiate I(GABA). Wild-type and mutant α(1)ß(2)γ(2) GABA(A)Rs were expressed in Xenopus laevis oocytes and analyzed using a two-electrode voltage clamp. GABA EC(50), BZD EC(50), and BZD maximal potentiation were measured. These data, combined with previous radioligand binding data describing the mutations' effects on BZD apparent binding affinities (J Neurosci 28:3490-3499, 2008; J Med Chem 51:7243-7252, 2008), were used to distinguish residues within the BZD pocket that contribute to BZD efficacy and BZD binding. We identified six residues whose mutation altered BZD maximal potentiation of I(GABA) (BZD efficacy) without altering BZD binding apparent affinity, three residues whose mutation altered binding but had no effect on BZD efficacy, and four residues whose mutation affected both binding and efficacy. Moreover, depending on the BZD ligand, the effects of some mutations were different, indicating that the structural mechanisms underlying the ability of BZD ligands with divergent structures to potentiate I(GABA) are distinct.

Packing of the extracellular domain hydrophobic core has evolved to facilitate pentameric ligand-gated ion channel function.

Protein function depends on conformational flexibility and folding stability. Loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. Here, using experimental and computational approaches, we show that eukaryotic pentameric ligand-gated ion channels are characterized by loose packing of their extracellular domain ß-sandwich cores, and that loose packing contributes to their ability to rapidly switch from closed to open channel states in the presence of ligand. Functional analyses of GABA(A) receptors show that increasing the ß-core packing disrupted GABA-mediated currents, with impaired GABA efficacy and slowed GABA current activation and desensitization. We propose that loose packing of the hydrophobic ß-core developed as an evolutionary strategy aimed to facilitate the allosteric mechanisms of eukaryotic pentameric ligand-gated ion channels.

Allosteric modulators induce distinct movements at the GABA-binding site interface of the GABA-A receptor.

Benzodiazepines (BZDs) and barbiturates exert their CNS actions by binding to GABA-A receptors (GABARs). The structural mechanisms by which these drugs allosterically modulate GABAR function, to either enhance or inhibit GABA-gated current, are poorly understood. Here, we used the substituted cysteine accessibility method to examine and compare structural movements in the GABA-binding site interface triggered by a BZD positive (flurazepam), zero (flumazenil) and negative (3-carbomethoxy-4-ethyl-6,7-dimethoxy-ß-carboline, DMCM) modulator as well as the barbiturate pentobarbital. Ten residues located throughout the GABA-binding site interface were individually mutated to cysteine. Wild-type and mutant α(1)ß(2)γ(2) GABARs were expressed in Xenopus laevis oocytes and functionally characterized using two-electrode voltage clamp. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive methanethiosulfonate (MTS) reagents in the absence and presence of BZD-site ligands and pentobarbital. Flurazepam and DMCM each accelerated the rate of reaction at α(1)R131C and slowed the rate of reaction at α(1)E122C, whereas flumazenil had no effect indicating that simple occupation of the BZD binding site is not sufficient to cause movements near these positions. Therefore, BZD-induced movements at these residues are likely associated with the ability of the BZD to modulate GABAR function (BZD efficacy). Low, modulating concentrations of pentobarbital accelerated the rate of reaction at α(1)S68C and ß(2)P206C, slowed the rate of reaction at α(1)E122C and had no effect at α(1)R131C. These findings indicate that pentobarbital and BZDs induce different movements in the receptor, providing evidence that the structural mechanisms underlying their allosteric modulation of GABAR function are distinct.

Disulphide trapping of the GABA(A) receptor reveals the importance of the coupling interface in the action of benzodiazepines.

BACKGROUND AND SIGNIFICANCE: Although the functional effects of benzodiazepines (BZDs) on GABA(A) receptors have been well characterized, the structural mechanism by which these modulators alter activation of the receptor by GABA is still undefined. EXPERIMENTAL APPROACH: We used disulphide trapping between engineered cysteines to probe BZD-induced conformational changes within the γ2 subunit and at the α1/γ2 coupling interface (Loops 2, 7 and 9) of α1ß2γ2 GABA(A) receptors. KEY RESULTS: Crosslinking γ2 Loop 9 to γ2ß-strand 9 (via γ2 S195C/F203C and γ2 S187C/L206C) significantly decreased maximum potentiation by flurazepam, suggesting that modulation of GABA-induced current (I(GABA)) by flurazepam involves movements of γ2 Loop 9 relative to γ2ß-strand 9. In contrast, tethering γ2ß-strand 9 to the γ2 pre-M1 region (via γ2S202C/S230C) significantly enhanced potentiation by both flurazepam and zolpidem, indicating γ2S202C/S230C trapped the receptor in a more favourable conformation for positive modulation by BZDs. Intersubunit disulphide bonds formed at the α/γ coupling interface between α1 Loop 2 and γ2Loop 9 (α1D56C/γ2L198C) prevented flurazepam and zolpidem from efficiently modulating I(GABA) . Disulphide trapping α1 Loop 2 (α1D56C) to γ2ß-strand 1 (γ2P64C) decreased maximal I(GABA) as well as flurazepam potentiation. None of the disulphide bonds affected the ability of the negative modulator, 3-carbomethoxy-4-ethyl-6,7-dimethoxy-ß-carboline (DMCM), to inhibit I(GABA) . CONCLUSIONS AND IMPLICATIONS: Positive modulation of GABA(A) receptors by BZDs requires reorganization of the loops in the α1/γ2 coupling interface. BZD-induced movements at the α/γ coupling interface likely synergize with rearrangements induced by GABA binding at the ß/α subunit interfaces to enhance channel activation by GABA.

The short splice variant of the gamma 2 subunit acts as an external modulator of GABA(A) receptor function.

GABA(A) receptors (GABA(A)Rs) regulate the majority of fast inhibition in the mammalian brain and are the target for multiple drug types, including sleep aids, anti-anxiety medication, anesthetics, alcohol, and neurosteroids. A variety of subunits, including the highly distributed gamma2, allow for pharmacologic and kinetic differences in particular brain regions. The two common splice variants gamma2S (short) and gamma2L (long) show different patterns of regional distribution both in adult brain and during the course of development, but show few notable differences when incorporated into pentameric receptors. However, results presented here show that the gamma2S variant can strongly affect both GABA(A)R pharmacology and kinetics by acting as an external modulator of fully formed receptors. Mutation of one serine residue can confer gamma2S-like properties to gamma2L subunits, and addition of a modified gamma2 N-terminal polypeptide to the cell surface recapitulates the pharmacological effect. Thus, rather than incorporation of a separate accessory protein as with voltage-gated channels, this is an example of an ion channel using a common subunit for dual purposes. The modified receptor properties conferred by accessory gamma2S have implications for understanding GABA(A)R pharmacology, receptor kinetics, stoichiometry, GABAergic signaling in the brain during development, and altered function in disease states such as epilepsy.

Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected.

The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA.

The barbiturate pentobarbital binds to gamma-aminobutyric acid type A (GABA(A)) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (approximately 10-100 microM), activate (approximately 100-800 microM), or block (approximately 1-10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABA(A) receptor, we labeled cysteine mutant receptors expressed in Xenopus oocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (F(TMRM)) of labels tethered to two residues in the extracellular domain (alpha(1)L127C and beta(2)L125C) that have been shown previously to produce an increase in F(TMRM) in response to GABA. Label at beta(2)K274C in the extracellular end of the M2 transmembrane helix reported a small but significant F(TMRM) increase during application of low modulating pentobarbital concentrations, and it showed a much greater F(TMRM) increase at higher concentrations. In contrast, GABA decreased F(TMRM) at this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels at alpha(1)L127C and beta(2)K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change in F(TMRM) at these sites, and the pattern of F(TMRM) signals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or activation by agonists, the structural changes in the GABA(A) receptor protein differ during transitions that are functionally equivalent.

Structural requirements for eszopiclone and zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) receptor are different.

The sleep-aids zolpidem and eszopiclone exert their effects by binding to and modulating gamma-aminobutyric acid type-A receptors (GABA(A)Rs), but little is known about the structural requirements for their actions. We made 24 cysteine mutations in the benzodiazepine (BZD) binding site of alpha(1)beta(2)gamma(2) GABA(A)Rs and measured zolpidem, eszopiclone, and BZD-site antagonist binding. Mutations in gamma(2)loop D and alpha(1)loops A and B altered the affinity of all ligands tested, indicating that these loops are important for BZD pocket structural integrity. In contrast, gamma(2)loop E and alpha(1)loop C mutations differentially affected ligand affinity, suggesting that these loops are important for ligand selectivity. In agreement with our mutagenesis data, eszopiclone docking yielded a single model stabilized by several hydrogen bonds. Zolpidem docking yielded three equally populated orientations with few polar interactions, suggesting that unlike eszopiclone, zolpidem relies more on shape recognition of the binding pocket than on specific residue interactions and may explain why zolpidem is highly alpha(1)- and gamma(2)-subunit selective.

A conserved salt bridge critical for GABA(A) receptor function and loop C dynamics.

Chemical signaling in the brain involves rapid opening and closing of ligand gated ion channels (LGICs). LGICs are allosteric membrane proteins that transition between multiple conformational states (closed, open, and desensitized) in response to ligand binding. While structural models of cys-loop LGICs have been recently developed, our understanding of the protein movements underlying these conformational transitions is limited. Neurotransmitter binding is believed to initiate an inward capping movement of the loop C region of the ligand-binding site, which ultimately triggers channel gating. Here, we identify a critical intrasubunit salt bridge between conserved charged residues (betaE153, betaK196) in the GABA(A) receptor (GABA(A)R) that is involved in regulating loop C position. Charge reversals (E153K, K196E) increased the EC(50) for GABA and for the allosteric activators pentobarbital (PB) and propofol indicating that these residues are critical for channel activation, and charge swap (E153K-K196E) significantly rescued receptor function suggesting a functional electrostatic interaction. Mutant cycle analysis of alanine substitutions indicated that E153 and K196 are energetically coupled. By monitoring disulfide bond formation between cysteines substituted at these positions (E153C-K196C), we probed the mobility of loop C in resting and ligand-bound states. Disulfide bond formation was significantly reduced in the presence of GABA or PB suggesting that agonist activation of the GABA(A)R proceeds via restricting loop C mobility.

Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions.

Gamma-aminobutyric acid (GABA) binding to GABA(A) receptors (GABA(A)Rs) triggers conformational movements in the alpha(1) and beta(2) pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA(A)R channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA(A)R pre-M1 regions. Alpha(1)beta(2) GABA(A)Rs containing cysteine substitutions in the pre-M1 alpha(1) (K219C, K221C) and beta(2) (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC(50) values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto alpha(1)K219C and alpha(1)K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For alpha(1)K221Cbeta(2) receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For alpha(1)beta(2)K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.

Structural mechanisms underlying benzodiazepine modulation of the GABA(A) receptor.

Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABA(A) receptors and modulating their function. The structural mechanisms by which BZD binding is transduced to potentiation or inhibition of GABA-induced current (I(GABA)) are essentially unknown. Here, we explored the role of the gamma(2)Q182-R197 region (Loop F/9) in the modulation of I(GABA) by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy-beta-carboline (DMCM)] BZD ligands. Each residue was individually mutated to cysteine, coexpressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes, and analyzed using two-electrode voltage clamp. Individual mutations differentially affected BZD modulation of I(GABA). Mutations affecting positive modulation span the length of this region, whereas gamma(2)W183C at the beginning of Loop F was the only mutation that adversely affected DMCM inhibition. Radioligand binding experiments demonstrate that mutations in this region do not alter BZD binding, indicating that the observed changes in modulation result from changes in BZD efficacy. Flurazepam and zolpidem significantly slowed covalent modification of gamma(2)R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that gamma(2)Loop F is a specific transducer of positive BZD modulator binding. Therefore, gamma(2)Loop F plays a key role in defining BZD efficacy and is part of the allosteric pathway allowing positive BZD modulator-induced structural changes at the BZD binding site to propagate through the protein to the channel domain.

Agonist-, antagonist-, and benzodiazepine-induced structural changes in the alpha1 Met113-Leu132 region of the GABAA receptor.

The structural basis by which agonists, antagonists, and allosteric modulators exert their distinct actions on ligand-gated ion channels is poorly understood. We used the substituted cysteine accessibility method to probe the structure of the GABAA receptor in the presence of ligands that elicit different pharmacological effects. Residues in the alpha1 Met113-Leu132 region of the GABA binding site were individually mutated to cysteine and expressed with wild-type beta2 and gamma2 subunits in Xenopus laevis oocytes. Using electrophysiology, we determined the rates of reaction of N-biotinaminoethyl methaneth-iosulfonate (MTSEA-biotin) with the introduced cysteines in the resting (unliganded) state and compared them with rates determined in the presence of GABA (agonist), 4-[6-imino-3-(4-methoxyphenyl)pyridazin-1-yl]butanoic acid hydrobromide (SR-95531; antagonist), pentobarbital (allosteric modulator), and flurazepam (allosteric modulator). alpha1N115C, alpha1L117C, alpha1T129C, and alpha1R131C are predicted to line the GABA binding pocket because MTSEA-biotin modification of these residues decreased the amount of current elicited by GABA, and the rates/extents of modification were decreased both by GABA and SR-95531. Reaction rates of some substituted cysteines were different depending on the ligand, indicating that barbiturate- and GABA-induced channel gating, antagonist binding, and benzodiazepine modulation induce specific structural rearrangements. Chemical reactivity of alpha1E122C was decreased by either GABA or pentobarbital but was unaltered by SR-95531 binding, whereas alpha1L127C reactivity was decreased by agonist and antagonist binding but not affected by pentobarbital. Furthermore, alpha1E122C, alpha1L127C, and alpha1R131C changed accessibility in response to flurazepam, providing structural evidence that residues in and near the GABA binding site move in response to benzodiazepine modulation.

Structural determinants for high-affinity zolpidem binding to GABA-A receptors.

The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the United States (Rush, 1998). Similar to classic benzodiazepines (BZDs), zolpidem binds at the extracellular N-terminal alpha/gamma subunit interface of the GABA-A receptor (GABAR). However, zolpidem differs significantly from classic BZDs in chemical structure and neuropharmacological properties. Thus, classic BZDs and zolpidem are likely to have different requirements for high-affinity binding to GABARs. To date, three residues--gamma2Met57, gamma2Phe77, and gamma2Met130--have been identified as necessary for high-affinity zolpidem binding (Proc Natl Acad Sci USA 94:8824-8829, 1997; Mol Pharmacol 52:874-881, 1997). In this study, we used radioligand binding techniques, gamma2/alpha1 chimeric subunits (chi), site-directed mutagenesis, and molecular modeling to identify additional gamma2 subunit residues important for high-affinity zolpidem binding. Whereas alpha1beta2chi receptors containing only the first 161 amino-terminal residues of the gamma2 subunit bind the classic BZD flunitrazepam with wild-type affinity, zolpidem affinity is decreased approximately 8-fold. By incrementally restoring gamma2 subunit sequence, we identified a seven-amino acid stretch in the gamma2 subunit loop F region (amino acids 186-192) that is required to confer high-affinity zolpidem binding to GABARs. When mapped to a homology model, these seven amino acids make up part of loop F located at the alpha/gamma interface. Based on in silico zolpidem docking, three residues within loop F, gamma2Glu189, gamma2Thr193, and gamma2Arg194, emerge as being important for stabilizing zolpidem in the BZD binding pocket and probably interact with other loop F residues to maintain the structural integrity of the BZD binding site.