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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Charged residues in the alpha1 and beta2 pre-M1 regions involved in GABAA receptor activation.

For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABAA receptor alpha1 (K219, R220, K221) and beta2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type beta2 or alpha1 in Xenopus oocytes. Cysteine substitution of beta2R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto alpha1K219C, beta2K213C, and beta2K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of alpha1K219C, beta2K213C, and beta2K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified beta2K213C and beta2K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of beta2K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

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.

Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein.

GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of alpha, beta, and gamma subunits can be confounded by receptors expressed from alpha and beta subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha1 subunit linked to a beta2 subunit. Cotransfection of the gamma2 subunit with alphabeta-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the gamma subunit with nonlinked alpha1 and beta2 subunits. Similarly, expression of alpha subunits together with alphabeta-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of alpha with beta subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of alphabeta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha1beta2 and alpha1beta2gamma2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in alpha1beta2gamma2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of alphabetagamma receptors.

An arginine involved in GABA binding and unbinding but not gating of the GABA(A) receptor.

GABA(A) receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose-response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function. Here, we focused on a residue (beta2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of beta2-R207, we expressed alpha1beta2 and alpha1beta2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because beta2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA(A) receptor (GABA(A)R) ligand-binding domain predicts that beta2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the beta2Ile154-Asp163 region in GABA(A)R activation, each residue was individually mutated to cysteine and coexpressed with wild-type alpha1 subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC50 value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) K(I) value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that beta2Thr160 and beta2Asp163 are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of beta2Glu155 resulted in spontaneously open GABA(A)Rs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto beta2E155C closed the spontaneously open channels, suggesting that beta2Glu155 is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the beta2 Ile154-Asp163 region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.

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.

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.