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Relative Positioning of Classical Benzodiazepines to the γ2‑Subunit of GABAA Receptors Simon J. Middendorp,† Evelyn Hurni,† Matthias Schönberger,‡ Marco Stein,‡ Michael Pangerl,‡ Dirk Trauner,‡ and Erwin Sigel*,† †

Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland Department of Chemistry, Ludwig-Maximilians-Universität München and Center of Integrated Protein Science, 81377 Munich, Germany



S Supporting Information *

ABSTRACT: GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors. γ-Aminobutyric acid type A receptors (GABAA) receptors are the major inhibitory neurotransmitter receptors in the mammalian central nervous system. They are chloride-selective ion channels composed of five subunits.1,2 GABAA receptors belong along with the nicotinic acetylcholine receptor, the 5HT3-receptor and the glycine receptor to the cys-loop pentameric ligand-gated ion channel family (LGIC). We focused on the major GABAA receptor isoform, which consists of 2α1, 2β2, and γ2 subunits3−5 that are arranged pseudosymmetrically around a central chloride-selective pore.1,6 The subunit arrangement has been shown to be γβαβα anticlockwise when viewed from the synaptic cleft.7−9 GABAA receptors possess a high-affinity binding site for modulators, such as benzodiazepines,10 that is located at the α/γ subunit interface.11−14 Benzodiazepines are positive allosteric modulators that potentiate GABA induced current but do not directly result in appreciable channel opening. They have a large spectrum of clinical actions. There is hope to separate these diverse drug actions, by finding compounds that act subtype specifically. For this purpose, it is important to understand how benzodiazepines interact with amino acid residues in their binding pocket. In this study, the proximity-accelerated chemical coupling reaction (PACCR) approach was used, which is ultimately based on the cysteine-accessibility method.15 Briefly, cysteinemutated receptors are combined with chemically modified ligands that bear cysteine-reactive groups (in this case isothiocyanate, -NCS) in different positions of the molecule.16,17 A covalent reaction will take place when the −S atom of the cysteine residue is in proximity to the reactive carbon © 2014 American Chemical Society

atom of -NCS. So far, two main interaction points between ligand and receptor have been identified. NCS compound (also termed NCS) reacted with α1H101C and α1N102C indicating apposition of these residues to the −Cl atom of diazepam18,19 and 3-NCS compound (also termed 3-NCS) reacted with α1S205C and α1T206C indicating that the −H atom attached to −C-3 carbon is in proximity to those amino acid residues.20 The chemical structures of NCS and 3-NCS are shown in Figure 1. The above interactions have recently also been studied in GABAA receptors containing different α subunit isoforms.21,22 These two contact points of classical benzodiazepines with amino acid residues still allow rotational freedom of the ligand in the binding site. Knowledge of amino acid residues on the γ2 subunit contacting benzodiazepine ligands is expected to give more detailed information about the relative orientation of the ligands in their binding pocket. We provide here experimental data showing the relative positioning of ligands of the benzodiazepine binding site to the γ2 subunit. The cysteine mutants of three amino acid residues on the γ2 subunit (Y58C, N60C, and V190C) covalently reacted with different cysteine-modified benzodiazepines.



RESULTS AND DISCUSSION Positioning of Benzodiazepines in Their Binding Pocket. We were interested in the relative positioning of Received: March 10, 2014 Accepted: June 11, 2014 Published: June 11, 2014 1846

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For mutant receptors covalently reacting with either NCS, 3NCS or bn-NCS a detailed analysis of the binding properties was performed (Table 1). α1β2γ2N60C, α1β2γ2Y58C, and Table 1. Binding Affinities of the Covalently Reacting Mutant Receptorsa receptor α1β2γ2b α1β2γ2Y58C α1β2γ2N60C α1β2γ2V190C

Ro15-1788 (Kd) [nM] 0.70 3.49 0.76 0.94

± ± ± ±

0.11 0.95 0.25 0.11

flunitrazepam (Kd) [nM] 2.0 2580 7.6 4.5

± ± ± ±

0.4 710c 2.7 0.9

diazepam (Ki) [nM] 14.5 31500 85.3 36.3

± ± ± ±

3.7 8400 24.6 10.7

a

In all cases binding of [3H]-Ro15-1788 and of [3H]-flunitrazepam and displacement of [3H]-Ro15-1788 by diazepam were determined. The exception is binding of flunitrazepam to α1β2γ2Y58C receptors, which was determined by displacement of [3H]-Ro15-1788. Receptors were expressed in HEK293 cells, membranes harvested and subjected to a radioactive ligand binding assay. Individual curves were first fitted and normalized to the maximal binding and subsequently averaged. Mean ± SD of three experiments ist shown in which each point was determined in triplicate. bValues for wild type α1β2γ2 receptors have been described before.24 cKi value determined by displacement of [3H]-Ro15-1788

Figure 1. Chemical structure of diazepam, flunitrazepam, Ro15-1788, NCS, 3-NCS, and bn-NCS.

diazepam in the benzodiazepine binding pocket in GABAA receptors. As mammalian GABAA receptors are not crystallized so far we chose a chemical biology approach to this question. Apposition of reactive amino acid residues with reactive ligand atoms lead to a covalent reaction. These covalent reactions were studied at the level of radioactive ligand binding where a covalent reaction blocks reversible binding of ligands and at the level of function using 2-electrode voltage clamp in Xenopus laevis oocytes. At the functional level a covalent reaction resulted in a irreversible alteration of the GABA response or in case of antagonism to a block of modulation by reversible ligands. Binding Properties of Cysteine-Reactive Compounds. We have made use of three reactive derivatives of classical benzodiazepines all carrying a NCS-group in different positions, NCS, 3-NCS and bn-NCS. All these compounds are shown in Figure 1. The affinities for compounds NCS and 3-NCS at wild type α1β2γ2 GABAA receptors have previously been reported in literature.18,20 They both retained affinity for the benzodiazepine-binding site. For compound bn-NCS that was newly synthesized the Ki for the displacement of [3H]-Ro15-1788 amounted to 410 ± 59 nM (mean ± SD, n = 3). Binding Properties of Cysteine Mutant Receptors. Twelve amino acid residues (γ2Tyr58, γ2Val59, γ2Asn60, γ2Ser61, γ2Ile62 all located in loop G; γ2Arg144 located in loop E; γ2Glu189, γ2Val190, γ2Gly191, γ2Asp192, γ2Thr193 all located in loop F, and α1Lys155 located in loop B) suspected to be part of or near the benzodiazepine binding site were selected based on homology models23 and individually mutated to cysteine. γ2 and α1 mutant subunits were expressed together with α1β2 and β2γ2, respectively. These cysteine mutant receptors were first tested for [3H]-Ro15-1788 and [3H]flunitrazepam binding. The expression level of α1β2γ2R144C receptors was too low to study PACCR. For α1β2γ2S61C receptors no specific binding of [3H]-Ro15-1788 was detected (see below). Covalent reaction of α1β2γ2S61C was therefore investigated with [3H]-flunitrazepam. All other mutant receptors bound [3H]-Ro15-1788 with estimated affinities between 0.5 and 3.5 nM. Except for mutant receptors α1β2γ2Y58C [3H]-flunitrazepam affinities ranged from 2.8 to 54 nM. Thus, the mutations to cysteine with these exceptions did not compromise [3H]-Ro15-1788 and [3H]-flunitrazepam binding.

α1β2γ2V190C receptors bound [3H]-Ro15-1788 with high affinities. Binding studies of α1β2γ2Y58C mutant receptor indicated a more than 1000 fold and more than 2000 fold decrease of affinities for flunitrazepam and diazepam, respectively, when compared with wild type receptors. The affinity of α1β2γ2N60C receptors for flunitrazepam and diazepam was about 4 and 6 fold lower than in wild type receptors, respectively. The mutation in residue γ2Val190 had only little effect on flunitrazepam and diazepam affinity. Observations with α1β2γ2S61C Receptors. As mentioned above for α1β2γ2S61C receptors no specific binding of [3H]-Ro15-1788 was detected. α1β2γ2S61C receptors bound flunitrazepam with an estimated affinity of about 54 nM (data not shown). This is a surprising result as serine is in many respects very similar to cysteine and may indicate that the side chain of this residue forms a hydrogen bridge that is essential for Ro15-1788 binding whereas flunitrazepam binding is affected to a smaller extent. Irreversible Reaction of NCS and 3-NCS at the Binding Level. We first studied interaction of the above-mentioned residues with the established modified ligands NCS and 3-NCS. Membranes of mutant receptors were exposed to one of the reactive compounds. After extensive washing to remove noncovalently bound ligand residual binding was determined in the presence of [3H]-Ro15-1788. A covalent reaction of the reactive ligand with the cysteine residue in the binding pocket will occlude reversible binding of the radioactive ligand. In case no covalent reaction occurred, the binding site should still be available for reversible binding. As a measure for intactness of the compounds NCS was incubated with α1H101Cβ2γ2 receptors and 3-NCS with α1S205Cβ2γ2 receptors. Both compounds strongly reduced reversible binding similarly as previously published18,20 (Figure 2A). Cysteine mutants of all above-mentioned residues were investigated for a covalent reaction with NCS and 3-NCS (Figure 2A). While α1β2γ2Y58C and α1β2γ2N60C receptors showed evidence for a covalent reaction, wild type α1β2γ2 and mutant receptors α1β2γ2V59C, α1β2γ2S61C, α1β2γ2I62C, α1K155Cβ2γ2 showed more than 80% residual binding after 1847

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Figure 2. (A) Irreversible reaction of NCS and 3-NCS with wild type and mutant α1β2γ2Y58C-I62C and α1K155Cβ2γ2 receptors. (B) Irreversible reaction of NCS, 3-NCS, and bn-NCS with wild type and mutant α1β2γ2E189C-T193C receptors. α1 and γ2 mutant subunits were coexpressed by transient transfection in HEK-cells with β2γ2 and α1β2, respectively. Membranes were exposed to 30 μM NCS, 10 μM 3-NCS or 0.3 μM bn-NCS. Residual binding was probed with [3H]-Ro15-1788. Data are shown in % of an internal control where the reactive compound was absent. Each bar shows mean ± SD of three to five experiments, in which points were determined in triplicates.

treatment with 30 μM NCS or 10 μM 3-NCS. Besides this, cysteine mutants of residues 189−193 on the γ2 subunit did also not react with NCS or 3-NCS. Residual binding was always >80% (Figure 2B). Treatment of mutant receptors α1β2γ2N60C with NCS resulted in a strong decrease of residual binding to 30 ± 6% (mean ± SD, n = 5). This corresponds to a covalent reaction of about 70%. This reaction could be prevented when NCS was preincubated with 100 mM L-cysteine for 1 h (data not shown). Interestingly, mutant receptor α1β2γ2Y58C reacted with both reactive molecules, NCS and 3-NCS, derived from positive allosteric modulators bearing the reactive groups on the opposite site of the ligand. Incubation with NCS and 3-NCS strongly reduced reversible binding to 30 ± 7 and 15 ± 3% corresponding to about 70 and 85% covalent reaction, respectively (mean ± SD, n = 3) (Figure 2A). Inactivation of NCS with 100 mM L-cysteine for 1 h almost completely prevented covalent reaction with γ2Y58C containing receptors (data not shown). Treatment with inactivated 3-NCS was done at the functional level (see below). Irreversible Reaction of Bn-NCS at the Binding Level. Surprisingly, treatment of wild type receptors with bn-NCS resulted in a strong covalent reaction at higher concentrations of the reactive compound that was characterized by an EC50 of 3.3 ± 2.0 μM (mean ± SD, n = 4) (Figure 3). So far, we were not able to identify the residue(s) in question. Reaction of wild type receptors has been observed before with Imid-NCS a reactive derivative of the antagonist Ro15-1788.25 At 0.3 μM bn-NCS the covalent reaction of wild type receptors amounted to about 15%. This concentration was used to screen several cysteine mutant receptors for a significant increase of the covalent reaction. Based on homology models bn-NCS was suspected to possibly react with cysteine mutants of residues 189−193 on loop F of the γ2-subunit. Nevertheless, residues 200−212 on the α1-subunit and 58 and 60 on the γ2 subunit were also investigated. The above-mentioned cysteine mutant receptors were individually incubated with 0.3 μM bn-NCS. Mutant receptor α1β2γ2V190C displayed about 45% covalent reaction (Figure 2B). To determine the EC50 of the covalent reaction of bn-NCS with α1β2γ2V190C receptors a full

Figure 3. Concentration-dependent covalent reaction of bn-NCS with wild type α1β2γ2 and mutant α1β2γ2V190C receptors. Receptors were exposed to increasing concentrations of bn-NCS for 1 h on ice and then extensively washed. In a subsequent binding assay with [3H]Ro15-1788 as a radioactive ligand residual binding was determined. Residual binding was then converted to percentage of binding sites covalently reacted. Data are shown as mean ± SD of three (α1β2γ2V190C) and four (α1β2γ2) experiments where each point was determined in triplicates.

concentration dependence experiment was performed. The EC50 amounted to 0.18 ± 0.01 μM (mean ± SD, n = 3) and revealed thus a shift to the left by about 20-fold (p < 0.05) as compared to wild type receptors (3.3 ± 2.0 μM, mean ± SD, n = 4) (Figure 3). Except for mutant receptor α1β2γ2V190C none of the tested cysteine mutants displayed a pronounced increase in the covalent reaction when incubated with 0.3 μM bn-NCS compared with wild type receptors (Figure 2B, data of mutant receptors α1200−212C, γ2Y58C, and γ2N60C not shown). Irreversible Reaction Studied at the Functional Level. Cysteine mutant receptors that showed a covalent modification at the binding level were also characterized at the functional level. For this purpose α1β2γ2N60C, α1β2γ 2Y58C, and α1β2γ2V190C receptors were expressed in Xenopus oocytes. GABA concentration response curves where fitted with EC50 values of 50.6 ± 12.7 μM, 40.7 ± 14.8 μM and 54.1 ± 6.2 μM for α1β2γ2N60C, α1β2γ2Y58C and α1β2γ2V190C receptors, respectively (mean ± SD, n = 3). These values should be compared with the EC50 obtained from wild type α1β2γ2 1848

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receptors that amounted to 45.6 ± 3.9 μM (mean ± SD, n = 3). None of the mutant receptors differed significantly in the apparent affinity for channel gating by GABA from wild type receptors. Next, the effect of the covalent modification with the cysteine-reactive compound on the GABA induced current response was investigated. Figure 4 shows current traces

Figure 5. Functional analysis of the effect of 3-NCS on α1β2γ2Y58C GABAA receptors expressed in Xenopus oocytes. (A) GABA (EC1−3) was applied twice to ensure a reproducible response. After exposure to 10 μM 3-NCS for 3 min (arrow) and extensive washing GABA was applied again which resulted in a large stable increase in the response to GABA that could not be antagonized by Ro15-1788. These experiments were repeated independently four times using different oocytes. (B) After a stable response to GABA (EC1−3) (single bars) has been obtained 1 μM Ro15-1788 was coapplied with GABA and a small increase of the current amplitude was recorded.

Figure 4. Functional analysis of the effect of NCS on α1β2γ2N60C mutant GABAA receptors expressed in Xenopus oocytes. (A) GABA (EC1−3) (single bars) was applied repetitively to ensure a reproducible response. In oocytes that were not perfused with NCS coapplication of Diazepam with GABA resulted in a large increase of the current amplitude. (B) oocytes were exposed to 40 μM NCS for 4 min (arrow) and extensively washed. The same concentration of GABA was applied again which resulted in a stable increase in the GABAelicited current. Subsequently, 1 μM diazepam was coapplied with GABA and a small increase in the current amplitude was recorded. All experiments were carried out four times. (C) Quantification of residual diazepam potentiation obtained from α1β2γ2N60C receptors. Additional potentation elicited by 1 μM diazepam together with GABA (EC1−3) after NCS-treatment is expressed as a percentage of the potentiation observed in independent oocytes expressing the same mutant receptor without previous exposure to NCS. Bars show mean ± SD of four experiments.

surprising in view of the fact that this mutant receptor showed a strongly reduced affinity for flunitrazepam and diazepam. Subsequent coapplication of 1 μM Ro15-1788 with GABA did not significantly change the current amplitude (411 ± 42%) (mean ± SD, n = 4). The high affinity antagonist Ro15-1788 could therefore not counteract the effect indicating a covalent reaction. In control experiments coapplication of 1 μM Ro151788 with GABA resulted only in a small increase of the current amplitude of 33 ± 8% (mean ± SD, n = 4) in naive oocytes (Figure 5B). In a protection experiment the covalent modification by 3NCS was inhibited by the presence of 10 μM Ro15-1788 1 min before, during and after exposure to 3-NCS. The residual irreversible potentiation of the GABA current in the presence of Ro15-1788 amounted to 27 ± 3% (mean ± SD, n = 4, p < 0.0001) of the potentiation observed in the absence of Ro151788 (Figure 6A). After preincubation of 3-NCS with 10 mM L-cysteine, which leads to prereaction of 3-NCS the stable increase in the GABA response was reduced to 3 ± 1% (mean ± SD, n = 4) of the potentiation recorded after 3-NCS treatment in the absence of L-cysteine. Treatment of oocytes expressing α1β2γ2Y58C receptors with 40 μM NCS resulted in an irreversible potentiation of the GABA response by 15 ± 8% (mean ± SD, n = 4), respectively. To test whether the binding site is occluded by reaction with NCS, we tested the allosteric properties of the receptor. Since diazepam failed to potentiate α1β2γ2Y58C receptors, the imidazopyridine zolpidem was used to demonstrate a covalent reaction. A subsequent coapplication of 3 μM zolpidem with GABA resulted in a current potentiation of 76 ± 32% (mean ±

obtained from oocytes expressing α1β2γ2N60C receptors. Perfusion of 40 μM NCS resulted in a stable increase of the current amplitude elicited by GABA that amounted to 28 ± 4% (mean ± SD, n = 4, Figure 4B). Subsequently, 1 μM diazepam was coapplied with GABA which led to a small current potentiation of 35 ± 11% (mean ± SD, n = 4, Figure 4B). This additional diazepam potentiation represented 24 ± 7% of potentation obtained from oocytes not treated with NCS (147 ± 12%, p < 0.0001) (Figure 4A). We estimate that about 76% of the sites were occluded by reacting with NCS (Figure 4C). Exposure of mutant α1β2γ2Y58C receptors to 3-NCS resulted in a large irreversible potentiation of the GABA elicited response, amounting to 386 ± 46% (mean ± SD, n = 4) (Figure 5A). This current potentiation was stable to prolonged perfusion with medium. This degree of potentiation is 1849

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type receptors at the binding level, these receptors were first investigated. A possible covalent reaction was probed with zolpidem. After exposure to 3 μM bn-NCS for 2 min the potentitation by 3 μM zolpidem in wild type α1β2γ2 receptors was not significantly reduced (379 ± 87%) (mean ± SD, n = 5). In α1β2γ2V190C receptors treatment with 3 μM bn-NCS significantly decreased zolpidem potentiation to 30 ± 6% (mean ± SD, n = 5, p < 0.01). This value should be compared to the potentiation of mutant receptors by zolpidem without treatment with bn-NCS which amounts to 52 ± 12% (n = 5). Novel Interaction Points Between Ligand and Receptor Identified in This Study. Out of 21 investigated cysteine mutant receptors,18,20 a prominent covalent reaction has so far been observed with α1H101C and α1N102C in the case of NCS and α1S205C and α1T206C in the case of 3-NCS. We describe here more contact points between ligand and receptor, especially in the γ2 subunit. As cysteine-reactive ligands NCS, 3-NCS and bn-NCS three NCS-modified derivatives of classical benzodiazepines were used. Bn-NCS is a new derivative bearing the NCS-group at a benzyl group that is attached to the nitrogen atom of the amide of the benzodiazepine molecule. The results are summarized in Table 2. NCS and 3-NCS were mainly investigated for additional covalent reactions with cysteine mutants of residues on loop G on the γ2 subunit. In chimera studies, it has been shown that residues Met57-Ile62 on loop G, in particular, Met57 and Tyr58, are essential determinants for conferring high-affinity binding of flunitrazepam.26 Furthermore, residue Met57 has been identified to be important for binding of zolpidem.27 The region of loop G most likely establishes a beta-sheet with residues Tyr58 and Asn60 pointing toward the binding site. Both corresponding cysteine mutations resulted in a reaction with NCS-modified benzodiazepines. As expected, neighboring residues of Tyr58 and Asn60, Val59 and Ser61 did not react with any NCS-derivative indicating that they most probably are pointing out of the pocket. Neighboring residues were suspected to be located too far away from the ligand and were therefore not investigated. Treatment of α1β2γ2Y58C receptors with 3-NCS resulted in a strong covalent reaction at the binding and at the functional level. The reaction could be suppressed by Ro15-1788 or prevented by preincubation with L-cysteine. These results therefore imply apposition of the hydrogen atom attached to −C-3 to residue γ2Tyr58. The functional results of α1β2γ2Y58C receptors with 3-NCS impressively demonstrate that despite of the low affinity of this mutant receptor to flunitrazepam and diazepam the ability to allosterically potentiate the GABA elicited response remains intact.

Figure 6. (A) Protection and cysteine-inactivation of the irreversible modulation by 3-NCS compound with α1β2γ2Y58C receptors expressed in Xenopus oocytes. GABA EC1−3 was applied before and after treatment with 10 μM 3-NCS for 1 min in the absence and presence of 10 μM Ro15-1788. The irreversible potentiation of the GABA current obtained with 10 μM 3-NCS in the presence of 10 μM Ro15-1788 was standardized to the one without. Additionally, covalent reaction of 3-NCS was prevented by preincubation of the compound with 10 mM L-cysteine for 1 h. Mean ± SD is shown from four experiments. (B) Quantification of residual zolpidem potentiation obtained from α1β2γ2Y58C receptors expressed in Xenopus oocytes. Additional potentation elicited by 3 μM zolpidem together with GABA (EC1−3) after NCS-treatment is expressed as a percentage of the potentiation observed in independent oocytes expressing the same mutant receptor without previous exposure of the NCS-compound. Bars show mean ± SD of four to seven experiments. Zolpidem potentiation after NCS-treatment was significantly reduced compared to potentiation obtained from untreated oocytes (p < 0.05).

SD, n = 5) (Figure 6B) for NCS-treated oocytes. This additional zolpidem potentiation represented 52 ± 22% of potentation obtained from oocytes not treated with NCS (146 ± 61%) (mean ± SD, n = 7, p < 0.05) indicating a covalent reaction. Covalent reaction of bn-NCS with α1β2γ2V190C receptors was also studied at the functional level. As a first step the reversible allosteric modulation by diazepam and zolpidem was investigated and compared with wild type α1β2γ2 receptors. Potentiation by 1 μM diazepam was reduced from 262 ± 27 (n = 3) in wild type receptors to only 53 ± 10 (n = 3) (p < 0.001) in α1β2γ2V190C receptors and by 3 μM zolpidem from 412 ± 36% (n = 5) to 52 ± 12% (n = 5) (p < 0.001). Next, the covalent reaction was studied. Since bn-NCS reacted with wild Table 2. Summary of the Novel Covalent Interactions Identified NCS

3-NCS

bn-NCS

receptor

binding level

function level

binding level

function level

binding level

function level

α1β2γ2 α1β2γ2Y58C α1β2γ2N60C α1β2γ2V190C

no reaction ∼70% ∼70% no reaction

no reaction reaction reaction

no reaction ∼85% no reaction no reaction

no reaction reaction

3.3 ± 2.0a no reaction no reaction 0.18 ± 0.01a

no reactionb

d

d d

d d

reactionc

a EC50 values for the concentration-dependent covalent reaction in μM. bAfter treatment of receptors with 3 μM bn-NCS, potentiation by 3 μM zolpidem was not significantly reduced. cAfter treatment of receptors with 3 μM bn-NCS, potentiation by 3 μM zolpidem was significantly reduced. d Experiments not done.

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Additionally, α1β2γ2Y58C receptors reacted with NCS both at the binding and at the functional level. The extent of the covalent reaction at the functional level was difficult to determine. Due to the low affinity of diazepam, zolpidem was used. Upon covalent reaction we observed a significant decrease of zolpidem potentiation, as compared to the potentiation obtained from untreated oocytes. This indicates that at least a part of the binding sites covalently bound NCS and became inaccessible for the positive allosteric modulator zolpidem. Residue γ2Tyr58 therefore has to be located relatively to the ligand in a way that the mutated residue can be reached by both NCS and 3-NCS. As mentioned before the region of loop G, where residues Tyr58-Ile62 are located, is predicted to establish a β-sheet. Residue Asn60 is therefore the residue that lies adjacent to Tyr58 pointing toward the binding pocket. The cysteine mutant of residue Asn60 reacted with NCS at both binding and functional level indicating direct apposition to the Cl-atom of diazepam. Besides γ2Y58C and γ2N60C, covalent reaction of bn-NCS was investigated with the cysteine mutants of residues γ2Glu189-Thr193 located on loop F and α1Ile201-Met212 located on loop C. We only observed a covalent reaction in the case of γ2V190C. Our results indicate a close apposition of the reactive entity in bn-NCS with residue γ2Val190. Due to the bulky nature of bn-NCS, this does not necessarily imply a direct contact of diazepam with this residue but results help to orient the ligand in the binding pocket. Implication for Modeling. Modeling of the relative positioning of benzodiazepines in their binding pocket has a long history.28−32 As far as it can be seen on 2d representations, none of the models predicts simultaneous access of γ2Y58C to −Cl and the hydrogen atom attached to −C-3 of diazepam, except the one by Xie et al.,32 that contrary to all other models and experimental evidence18 places α1His101 very distant from the position corresponding to −Cl in diazepam. Two studies were based largely on docking,28,32 which in the absence of experimental data is difficult to interpret. Three studies that took into account the two ligand attachment points α1His101, and α1Ser205/α1Thr206 have resulted in conflicting data.29−31 Reasons are the pseudosymmetry of the ligand relative to the hydrophobic regions and the fact that two interactions still allow rotational freedom of the ligand in the binding site. A model should be verified by prediction of novel ligands. For all these modeling studies prediction of novel ligands was either not performed or had limited success. New hope has been provided with the crystallization of homologous proteins of the GABAA receptor such as ELIC,33,34 GLIC35,36 and GluCl37 with a higher total similarity than the nAChR38 and AChBP.39 However, they also lack the benzodiazepine binding site. Summary and Conclusions. In the absence of a crystal structure of the ligand-bound benzodiazepine binding site of GABAA receptors PACCR is a powerful approach to determine the architecture and the spatial representation of the benzodiazepine binding pocket. The herein provided data about the relative positioning of the γ2-subunit to the ligand strongly improve the knowledge about the three-dimensional orientation of ligands in the benzodiazepine binding site. The ligand is now locked in a certain orientation without rotational freedom. To summarize these findings, a new scheme is proposed that includes the three amino acid residues on the γ2 subunit (Figure 7). α1His101, α1Asn102, and γ2Asn60 cluster around the Cl-atom of diazepam. α1Ser205 and α1Thr206 are

Figure 7. Schematic representation of the relative positioning of diazepam in the benzodiazepine binding pocket. Residues α1His101, α1Asn102, α1Ser205, and α1Thr206 have been identified previously.18−20 Residues of which the cysteine mutation resulted in a covalent reaction with NCS are shown in red, with 3-NCS in blue, with bn-NCS in green and with both NCS and 3-NCS in orange. Residues Asn60 and Tyr58 are located above the ligand as judged by available homology models.13

apposed to the −H atom attached to −C-3. Reaction of γ2Y58C with both NCS and 3-NCS implies localization of this residue between the −Cl and the −H attached to −C-3 of diazepam. Val190 is located in the region of the N-methyl of diazepam. So far, we identified a total of 8 covalent reactions. These experimental data have been used for the generation of a pharmacophore. This pharmacophore has been used for a prospective search of novel ligands. A receptor-based virtual screening resulted in 14 lead compounds of different scaffolds with an affinity

Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site a...
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