Conformational changes at benzodiazepine binding sites of GABA(A) receptors detected with a novel technique

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA(A) receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, alpha1H101Cbeta2gamma2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the alpha/gamma subunit interface, or if it occurs in both alpha1 subunits, exclusively reaction at the alpha/gamma subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 microm GABA but only partially in its absence or in the presence of 10 microm GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.     

Deciphering the binding mode of Zolpidem to GABA<Subscript>A</Subscript> α<Subscript>1</Subscript> receptor – insights from molecular dynamics simulation

To investigate the binding mode of Zolpidem to GABA(A) and to delineate the conformational changes induced upon agonist binding, we carried out atomistic molecular dynamics simulation using the ligand binding domain of GABA(A) α(1) receptor. Comparative molecular dynamics simulation of the apo and the holo form of GABA(A) receptor revealed that γ(2)/α(1) interface housing the benzodiazepine binding site undergoes distinct conformational changes upon Zolpidem binding. We notice that C loop of the α(1) subunit experiences an inward motion toward the vestibule and the F loop of γ(2) sways away from the vestibule, an observation that rationalizes Zolpidem as an alpha1 selective agonist. Energy decomposition analysis carried out was able to highlight the important residues implicated in Zolpidem binding, which were largely in congruence with the experimental data. The simulation study disclosed herein provides a meaningful insight into Zolpidem-GABA(A)R interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.     

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

Action of analogues on γ-aminobutyric acid recognition sites in rat brain

With a view to finding potential GABA-mimetics, the effects of a number of structural analogues of GABA were studied on three parameters associated with GABA neural transmission of rat brain. These were (1) the binding of [³H]GABA to its receptor, (2) the binding of [³H]GABA to its transporter (sodium-dependent binding), and (3) the activity of GABA aminotransferase. Thirteen of the 21 compounds tested competitively inhibited both the low and the high affinity GABA receptor binding components. The most potent inhibitors were morpholinopropane sulphonic acid (MOPS) and aminoethylthiosulphonic acid (AETS). All of the compounds were markedly less effective in inhibiting the high affinity GABA receptor binding system than the low affinity system. The effect of each of the inhibitors was measured on [³H]diazepam receptor binding. Only 6-(morpholinomethyl)kojic acid, kojic amine, 1-piperidinepropane sulphonic acid and 4(4′-azido-benzoimidylamino)butanoic acid (ABBA) were able to induce a stimulation of binding. Four of the inhibitors of [³H]GABA binding were able to appreciably reduce GABA-induced enhancement of diazepam binding. These were N-(2-nitro,4-azidophenyl)aminopropane sulphonic acid, 8-amino-1-napthalene sulphonic acid, narcotine-N-oxide and 5-phenyl-2-pyrrolepropionic acid. These results demonstrate that MOPS and AETS are good inhibitors of GABA receptor binding although there is no other evidence that they might be agonists since they have no effect on diazepam receptor binding. Based on their ability to block GABA-induced stimulation of diazepam binding ABBA, 8-amino-1-naphthalene sulphonic acid and 5-phenyl-2-pyrrolepropionic acid may possess antagonistic properties. ABBA was the only compound to inhibit sodium-dependent [³H]GABA binding. None of the compounds had an effect on the activity of GABA aminotransferase. From this study at least two analogues, MOPS and AETS, have emerged that hold potential as GABA-mimetics. Also, the three GABA recognition sites of rat brain have been shown to possess marked pharmacological differences.     

Identification of the bovine gamma-aminobutyric acid type A receptor alpha subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513

Ligands binding to the benzodiazepine-binding site in gamma-aminobutyric acid type A (GABA(A)) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABA(A) receptor alpha and gamma subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [(3)H]flunitrazepam identified a primary site of incorporation at alpha(1)His-102, whereas studies with [(3)H]Ro15-4513 suggested incorporation into the alpha(1) subunit at unidentified amino acids C-terminal to alpha(1)His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified ( approximately 200-fold) GABA(A) receptor from detergent extracts of bovine cortex, photolabeled it with [(3)H]Ro15-4513, and identified (3)H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of (3)H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different alpha subunits. Based upon this radiochemical sequence analysis, [(3)H]Ro15-4513 was found to selectively label the homologous tyrosines alpha(1)Tyr-210, alpha(2)Tyr-209, and alpha(3)Tyr-234, in GABA(A) receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure.     

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.     

GABA agonists

Summary This review describes the development of GABA receptor agonists with no detectable affinity for other recognition sites in GABA-mediated synapses. The key compounds are THIP, isoguvacine, and piperidine-4-sulphonic acid (P4S), developed via extensive structural modifications of the potent but not strictly specific GABA agonist muscimol. The structural parameters, which have to be considered in the design of GABA agonists are discussed on the basis of the structures and biological activities of these GABA agonists and a number of related compounds.A model, which summarizes our present knowledge of the structure of the postsynaptic GABA receptor complex, is presented, and the interaction of GABA agonists with various sites in this complex is discussed. Of particular interest are the effects of GABA agonists on the binding of diazepam to the benzodiazepine binding site, assumed to be a structural unit of the GABA receptor complex. While rigid molecules like THIP are capable of activating the GABA receptors, a certain degree of conformational mobility of GABA agonists apparently is a prerequisite for stimulation of diazepam binding in vitro at 0 °C. These findings suggest that GABA receptor functions involve conformational changes of certain elements of the receptor complex.Some aspects of the pharmacology of GABA agonists are discussed, including the attempts to develop GABA agonists with desirable pharmacokinetic and toxicological characteristics. While muscimol is a toxic compound, THIP is well tolerated by animals, and in contrast to isoguvacine, THIP penetrates into the brain after systemic administration to animals, a difference which can be explained on the basis of their protolytic properties. The attempts to develop pro-drugs of isoguvacine capable of penetrating the blood-brain barrier with subsequent decomposition in the brain tissue to isoguvacine are described.     

GABA-benzodiazepine interactions: physiological, pharmacological and developmental aspects

Many of the pharmacological actions of the benzodiazepines can be attributed to their actions on gamma-aminobutyric acid (GABA) systms in the brain. Electrophysiological studies on dorsal raphe neurons indicate that the benzodiazepines act postsynaptically to potentiate GABAergic inhibition in this midbrain nucleus. Direct binding studies have shown that both in vitro and in vivo binding of [3H]diazepam to a specific high affinity benzodiazepine binding site in cerebral cortical tissue are enhanced by the direct in vitro addition of GABA and GABA agonists or by pretreatment of animals with GABA analogs and agents that elevate GABA levels in brain. Ontogenic development of [3H]diazepam binding in brain parallels the development of the sodium-independent [3H]GABA binding. The ability of GABA to enhance benzodiazepine binding is present throughout development and inversely related to age. These data suggest that there is a functionally significant interaction between the benzodiazepines and GABA throughout development and at maturity. A model is proposed to relate these interactions to conformational changes in a benzodiazepine/GABA/Cl- ionophore complex.     

Characterization of gamma-aminobutyric acid-benzodiazepine receptor complexes by protection against inactivation by group-specific reagents

Abstract: The chemical topography of the γ-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino- and tyrosyl-selective reagents and the protection from it by direct and allosteric ligands of the GABA-BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p -diazobenzenesulfonic acid (DSA), tetrani-tromethane (TNM), and N -acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABA_A site. TNM, like DSA, selectively decreased the number of the low-affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low-affinity sites. TNM pre-treatment also abolished the muscimol enhancement of [³H]diazepam binding, which suggests that the low-affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal-5-phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABA_A receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [³H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [³H]diazepam binding by N -bromosuccinimide and N -acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.     

Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.     

How to choose relevant multiple receptor conformations for virtual screening: a test case of Cdk2 and normal mode analysis

Better treatment of protein flexibility is essential in structure-based drug design projects such as virtual screening and protein-ligand docking. Diversity in ligand-binding mechanisms and receptor conformational changes makes it difficult to treat dynamic features of the receptor during the docking simulation. Thus, the use of pregenerated multiple receptor conformations is applied today in virtual screening studies. However, generation of a small relevant set of receptor conformations remains challenging. To address this problem, we propose a new protocol for the generation of multiple receptor conformations via normal mode analysis and for the selection of several receptor conformations suitable for docking/virtual screening. We validated this protocol on cyclin-dependent kinase 2, which possesses a binding site located at the interface between two subdomains and is known to undergo significant conformational changes in the active site region upon ligand binding. We believe that the suggested rules for the choice of suitable receptor conformations can be applied to other targets when dealing with in silico screening on flexible receptors.     

Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the β2-α1 interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent α1-β2 subunit interface. However, as antagonists and agonists produce identical α1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent β2 subunits in α1 β2 receptors can dimerize via K24'C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Together, this information provides new insights into the activation mechanisms of Cys-loop receptors.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

On the benzodiazepine binding pocket in GABAA receptors

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.