The GABAA Receptor alpha 1 Subunit Pro174-Asp191 Segment Is Involved in GABA Binding and Channel Gating*

J. Glen Newell and Cynthia CzajkowskiDagger

From the Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received for publication, November 21, 2002, and in revised form, January 14, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To define possible roles of the GABAA receptor alpha 1 subunit Pro174-Asp191 segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue was individually mutated to cysteine, expressed with wild-type beta 2 subunits in Xenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC50 values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in the KI values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation. Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed the modification of R186C, suggesting that this region of the alpha 1 subunit may act as a dynamic element during channel-gating transitions.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Allosteric transitions of neurotransmitter binding sites remain poorly understood, despite increased efforts in recent years to map protein domains important for ligand recognition and ion channel activation. It is likely that amino acid residues other than those that mediate initial contact with agonist will be important for inducing gating transitions. Characterization of receptor-ligand interactions using site-directed mutagenesis and photolabeling studies provides limited information as to the state-dependent nature of ligand binding domains (1). Nevertheless, identification of all residues lining the neurotransmitter binding-site, irrespective of the conformational state of the receptor, represents a critical step to understanding receptor-ligand interactions at allosteric proteins.

Identification of amino acid residues important in agonist/antagonist binding at gamma -aminobutyric acid type A receptors (GABAAR)1 reveals that the GABA-binding sites are located at beta -alpha subunit interfaces. Consistent with the agonist-binding site of nicotinic acetylcholine receptors (nAChR), the GABA-binding site is formed by amino acid residues clustered in non-continuous protein segments of the extracellular amino-terminal domains of adjacent subunits. Multiple residues have been implicated in the formation of this binding site using a variety of approaches, including site-directed mutagenesis, photoaffinity labeling, and the substituted-cysteine accessibility method (SCAM). These include Phe64, Arg66, Arg119, and Ile120 of the alpha 1 subunit (2-7), in addition to Tyr97, Leu99, Tyr157, Thr160, Thr202, Ser204, Tyr205, Arg207, and Ser209 of the beta 2 subunit (8-10). Of these residues, it is likely that some contact agonist/antagonist molecules directly, some maintain the overall structure of the binding site, while others mediate conformational dynamics within the site during allosteric transitions among the resting, active, and desensitized states.

The GABAAR alpha 1 subunit segment between Pro174 and Asp191 is homologous in position to the putative "loop F" of the nAChR (see Fig. 1) (11). Studies of this segment of the nAChR gamma /epsilon and delta  subunits have identified negatively charged amino acid residues that influence acetylcholine binding, channel gating, and perhaps potassium ion interactions (see Fig. 1) (12-16). Based on the crystal structure of a soluble acetylcholine-binding protein (AChBP), a protein homologous to the extracellular domain of the nAChR, the secondary structure of the loop F region is predicted to be a random coil (17). Strikingly, the loop F protein sequence is poorly conserved among all GABAAR subunit isoforms and other related ligand-gated ion channel subunits and may represent a unique structural element that could account for differences in agonist affinity, dimensions of binding pockets, and access pathways important for receptor-ligand interactions. Therefore, an analysis of the structure and the role(s) of the alpha 1 subunit Pro174-Asp191 segment in ligand binding and ion channel activation is fundamental for understanding GABAAR function.

The development of SCAM has proved to be very powerful tool for identifying residues important for the pharmacology of both agonists and antagonists. Originally developed to identify the channel-lining residues of ligand-gated ion channels (18), SCAM has gained widespread use in the study of the ligand binding domains of these channels (2, 3, 9, 10, 19-25). The method entails introduction of successive cysteine residues, one at a time, within a protein domain and expression of recombinant receptors in heterologous systems. Solvent accessibility of a given cysteine is determined by monitoring changes in function following application of a sulfhydryl-specific modifying reagent (18). The role of a given residue in the formation of a ligand binding site is determined by the ability of both agonists and antagonists to impede modification of the introduced cysteine by the sulfhydryl-specific reagent.

Here, we used SCAM to examine the structure, solvent accessibility, and dynamics of the GABAA receptor alpha 1 subunit Pro174-Asp191 region, which comprises the putative loop F of the GABA binding pocket. We demonstrate that this region is highly accessible and adopts a random coil/turn conformation. In addition, we identify several residues, Val178, Val180, and Asp183, that likely participate in forming part of the GABA-binding pocket. Moreover, we provide evidence that this region of the receptor undergoes conformational rearrangements during pentobarbital-mediated gating of the channel. The results are discussed in terms of a homology model of the GABAAR agonist binding site, based on the recently solved crystal structure of the AChBP (17).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Mutagenesis and Expression in Oocytes-- Rat cDNAs for the alpha 1 and beta 2 subunits of the GABAA receptor were used in this study. The alpha 1 cysteine mutants were engineered using a recombinant polymerase chain reaction method, as described previously (2, 10, 26). Cysteine substitutions were made in the alpha 1 subunit at Pro174, Ala175, Arg176, Ser177, Val178, Val179, Val180, Ala181, Glu182, Asp183, Gly184, Ser185, Arg186, Leu187, Asn188, Gln189, Tyr190, and Asp191 (Fig. 1). Cysteine substitutions were verified by restriction endonuclease digestion and double-stranded DNA sequencing.


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Fig. 1.   The Pro174-Asp191 segment (loop F) of the rat GABAAR alpha 1 subunit is aligned with analogous regions of the rat GABAAR beta 2 and gamma 2 subunits and rat nAChR gamma , epsilon , and delta  subunits. The numbering reflects the position of the residues in the mature GABAAR alpha 1 subunit. Residues implicated in acetylcholine binding are circled and include delta Asp180, delta Glu189, gamma Asp174, epsilon Asp175, and epsilon Asn182 (12-14, 16), while residues that line the GABA-binding site are boxed. Residues implicated in the interactions of divalent cations are underlined (15, 46). The asterisks (*) indicate gaps in the amino acid sequence alignment.

All wild-type and mutant cDNAs were subcloned into the vector pGH19 (27, 28) for expression in Xenopus laevis oocytes. Oocytes were prepared as previously described (29). cRNA transcripts were prepared using the T7 mMessage machine (Ambion). GABAA receptor beta 2 and alpha 1 or alpha 1 mutant subunits were co-expressed by injection of cRNA (200-800 pg/subunit) in a 1:1 ratio (alpha :beta ). The oocytes were maintained in ND96 medium (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2 and 5 HEPES, pH 7.4)and supplemented with 100 µg/ml gentamicin and 100 µg/ml bovine serum albumin. Oocytes were used 2-7 days after injection for electrophysiological recordings.

Two-electrode Voltage Clamp Analysis-- Oocytes under two-electrode voltage clamp were perfused continuously with ND96 at a rate of ~5 ml/min. The holding potential was -80 mV. The volume of the recording chamber was 200 µl. Standard two-electrode voltage clamp procedures were carried out using a GeneClamp500 Amplifier (Axon Instruments, Inc.). Borosilicate electrodes were filled with 3 M KCl and had resistances of 0.5-3.0 MOmega in ND96. Stock solutions of GABA (Sigma) and SR-95531 (Sigma) were prepared in water, while N-biotinylaminoethyl methanethiosulfonate (100 mM) (MTSEA-biotin, Biotium, Hayward, CA) was prepared in dimethyl sulfoxide (Me2SO). All compounds were prepared fresh daily and MTSEA-biotin was diluted appropriately in ND96 such that the final concentration of Me2SO was <= 2%. This solvent concentration did not affect recombinant GABAAR.

To measure the sensitivity to GABA, the agonist (0.0001-1 mM) was applied via gravity perfusion or by pipettor application (~5-8 s) with a 3-15-min washout period between each application to ensure complete recovery from desensitization. Peak GABA-activated current (IGABA) was recorded. To correct for slow drift in the maximum amplitude of the response as a function of time, concentration-response data were normalized to a low concentration of GABA (EC2-EC5). Concentration-response curves were generated for each recombinant receptor, and the data were fit by non-linear regression analysis using GraphPad Prism software (San Diego, CA; graphpad.com). Data were fit to the following equation I = Imax/(1 + (EC50/[A])n), where I is the peak amplitude of the current for a given concentration of GABA ([A]), Imax is the maximum amplitude of the current, EC50 is the concentration required for half-maximal receptor activation, and n is the Hill coefficient.

To measure the sensitivity to SR-95531, GABA (EC50) was applied via gravity perfusion followed by a brief (20 s) washout period before co-application of GABA (EC50) and increasing concentrations of SR-95531. The response to the application of SR-95531 and GABA was normalized to the response elicited by the agonist alone. Concentration-inhibition curves were generated for each recombinant receptor, and the data were fit by non-linear regression analysis using GraphPad Prism software. Data were fit to the following equation: 1 - 1/(1 + (IC50/[Ant])n), where IC50 is the concentration of antagonist ([Ant]) that reduces the amplitude of the GABA-evoked current by 50% and n is the Hill coefficient. KI values were calculated using the Cheng-Prussof correction: KI = IC50/(1 + ([A]/EC50)), where [A] is the concentration of GABA used in each experiment and EC50 is the concentration of GABA that elicits a half-maximal response for each receptor (30).

Modification of Introduced Cysteine Residues by MTSEA-biotin-- MTSEA-biotin was the sulfhydryl-specific reagent used in this study. It is a relatively impermeant compound (31) with dimensions (14.5 Å unreacted moiety; 11.2 Å reacted moiety) that are similar to SR-95531 (13.5 Å) but much longer than GABA (4.5 Å). Methanethiosulfonate reagents react 109-1010 times faster with the ionized thiolate (RS-) form of cysteine than the unionized form (32). Based on these properties, it is reasonable to assume that MTSEA-biotin can occupy the GABA-binding site and that this reagent will principally modify extracellular cysteine residues that are solvent-exposed.

Oocytes expressing either wild-type or mutant receptors were activated by GABA (EC50) at regular intervals until the peak current amplitude varied by <= 10% on two consecutive applications. Oocytes were then allowed to fully recover, after which a high concentration of MTSEA-biotin (2 mM) was applied (2 min). Following MTSEA-biotin application, cells were washed (5 min) with ND96, after which GABA (EC50) was again applied to determine the effect of MTSEA-biotin application on IGABA. The effect of MTSEA-biotin was calculated as the difference in the amplitude of the IGABA before and after MTSEA-biotin application as follows: (IGABApre - IGABApost/IGABApre) × 100, where post refers to the amplitude of IGABA following MTSEA-biotin application and pre refers to the amplitude of IGABA prior to exposure to MTSEA-biotin.

Rate of Modification of Introduced Cysteine Residues-- Rates were measured only for those cysteine mutants that had a >40% change in IGABA following MTSEA-biotin treatment (2 min, 2 mM). The rate at which MTSEA-biotin modified introduced cysteine residues was measured using low MTSEA-biotin concentrations as described previously (3). In general, the concentration of MTSEA-biotin used was 50 µM, with the exception of A181C (500 nM) and R186C (5 µM). The experimental protocol is described as follows: GABA (EC50) application (5 s); ND96 wash-out (25 s); MTSEA-biotin application (10-20 s); ND96 washout (2.2-2.3 min). The sequence was repeated until IGABA no longer changed following the MTSEA-biotin treatment (i.e. the control reaction had proceeded to apparent completion). The individual abilities of GABA, SR-95531, and pentobarbital to alter the rate of cysteine modification by MTSEA-biotin were determined by co-applying either GABA (5 × EC50), SR-95531 (40 × KI), or an activating concentration of pentobarbital (500 µM) during the MTSEA-biotin pulse. In all cases, the wash times were adjusted to ensure that currents obtained from test pulses of GABA (EC50) following exposure to high concentrations of GABA, SR-95531, or pentobarbital were stabilized. This ensured complete wash-out of drugs and that any reductions in the current amplitude were the result of MTSEA-biotin application.

For all rate experiments, the decrease in IGABA was plotted as a function of the cumulative time of MTSEA-biotin exposure and fit to a single-exponential decay function using GraphPad Prism software. A pseudo-first order rate constant (k1) was determined and the second order rate constant (k2) was calculated by dividing k1 by the concentration of MTSEA-biotin used in the assay (33). Second order rate constants were determined using at least two different concentrations of MTSEA-biotin.

Statistical Analysis-- log (EC50) and log (KI) values were analyzed using a one-way analysis of variance, followed by a post-hoc Dunnett's test to determine levels of significance between wild-type and mutant receptors. Differences among the second order (k2) rates of covalent modification of the various mutants were assessed using the false positive discovery rate method (34). This method limited the expected percent of false positives to 5%. The false positive discovery rate is a more meaningful measure of error in large screening experiments than the more traditional approach of limiting the probability of one or more false positives (also known as experiment-wise error control). Before analysis, the rates were transformed to a log scale to obtain more normally distributed residuals. Results are reported in the original scale. Even using this approach, clear trends in the data did not always achieve significance as has been noted in other large assays using SCAM (35).

Structural Modeling-- The mature protein sequences of the rat alpha 1 and beta 2 subunits were homology-modeled with a subunit of the AChBP (17). The crystal structure of the AChBP was downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (code 1I9B) and loaded into Swiss Protein Bank Viewer (SPDBV, ca.expasy.ord/spdbv). The alpha 1 protein sequence from Thr12-Ile227 and the beta 2 protein sequence from Ser10-Leu218 were aligned with the AChBP primary amino acid sequence as depicted in Cromer et al. (36) and threaded onto the AChBP tertiary structure using the "Interactive Magic Fit" function of SPDBV. The threaded subunits were imported into SYBYL (Tripos, Inc., St. Louis, MO) where energy minimization was carried out (<0.5 kcal/Å). The first 100 iterations were carried out using Simplex minimization (37) followed by 1000 iterations using the Powell conjugate gradient method (38). A beta 2/alpha 1 GABA-binding site interface was assembled by overlaying the monomeric subunits on the AChBP scaffold, the resulting structure was imported into SYBYL, and energy was minimized. Our model is quite similar to models recently published for the nAChR and GABAAR ligand binding domains (36, 39). It is worth noting that positioning of the alpha 1 subunit Pro174-Asp191 (loop F) region is inexact since the alpha 1 subunit has low homology to AChBP sequence in this region and contains three additional amino acid residues. This region and other regions with insertions were modeled by fitting structures from a loop data base.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Expression and Functional Characterization of GABAAR alpha 1 Subunit Cysteine Mutants-- Cysteine substitutions were engineered at eighteen individual positions in the GABAAR alpha 1 subunit (Pro174, Ala175, Arg176, Ser177, Val178, Val179, Val180, Ala181, Glu182, Asp183, Gly184, Ser185, Arg186, Leu187, Asn188, Gln189, Tyr190, and Asp191) and co-expressed with wild-type beta 2 subunits in X. laevis oocytes for functional analysis using the two-electrode voltage clamp method. Expression of most mutant subunits produced GABA-activated channels with the exceptions of L187C and Q189C (Fig. 2 and Table I). The lack of functional expression of receptors carrying the L187C and Q189C mutations may indicate a role for these residues in receptor synthesis/assembly as they are conserved in all GABAAR and glycine receptor subunits. Expression of D183C produced a significant 7-fold rightward shift in EC50 relative to wild-type values (EC50 = 1.6 µM). However, the KI values for the competitive antagonist, SR-95531, for mutant receptors were not significantly different from wild-type values (KI = 330 nM). Hill coefficients were not significantly different from wild type (Table I). In general, the maximum current amplitude was 1-10 µA for wild-type and mutant receptors, with the exception of R186C (<300 nA).


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Fig. 2.   A, concentration-response curves of GABA-activated current for wild-type (black-square) and recombinant alpha 1beta 2 receptors carrying the D183C mutation () expressed in Xenopus oocytes. Data were normalized to peak IGABA for each experiment. Data represent the mean ± S.E. of at least three independent experiments. B, concentration-dependence of SR-95531-mediated reduction of IGABA (EC50) for wild-type (black-square) and recombinant receptors carrying the D183C mutation (). Data represent the mean ± S.E. of at least three independent experiments. The EC50 values, KI values, and calculated Hill coefficients are summarized in Table I.


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Fig. 3.   Summary of the effects of MTSEA-biotin (2 mM) on wild-type and mutant receptors. A, representative current traces demonstrating the effects of MTSEA-biotin (2 mM) application on GABA-mediated current (EC50) at wild-type, and V178C-, V180C-, and A181C-containing receptors. The arrows in the current traces represent MTSEA-biotin application (2 min), and the breaks in the current trace represent the subsequent wash (5 min). B, summary of the maximum effect of MTSEA-biotin at all receptors. Effect is calculated as % change = ([IGABApost MTSEA-biotin/IGABApre MTSEA-biotin- 1) × 100. Results represent the mean ± S.E. for three to six experiments. The closed bars indicate values that were statistically different from wild-type values (p < 0.05). The pattern of accessibility suggests that this domain of the alpha 1 subunit forms a random turn/coil. Cysteine substitutions are positions 187 and 189 were not tolerated.


                              
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Table I
Concentration-response data for GABA activation and SR-95531 inhibition of wild-type and mutant receptors expressed in Xenopus oocytes
Data represent the mean ± S.E. for three to four experiments (n). Values for EC50 and Hill slopes (nH) were determined from concentration-response data using non-linear regression analysis with GraphPad Prism software. Hill slopes and log (EC50) values were analyzed using a one-way analysis of variance followed by a Dunnett's test to determine the levels of significance (*, p < 0.01).

These data suggest that cysteine substitution within this domain of the GABAAR alpha 1 subunit protein is well tolerated. A major assumption of SCAM is that the side chain of the introduced cysteine is in a similar position as the side chain of the native residue. Since GABA and SR-95531 bind equally well to both mutant and wild-type receptors, it is likely that the structures of the receptors are similar.

Modification of Introduced Cysteine Residues by MTSEA-biotin-- To define the surface accessibility of the alpha 1 subunit P174C-D191C segment, wild type and mutant receptors were exposed to MTSEA-biotin (2 mM) for 2 min (Fig. 3). MTSEA-biotin had no effect on wild-type receptors. MTSEA-biotin significantly reduced IGABA at P174C (60.5 ± 1.1%, n = 3), R176C (39.3 ± 6.3%, n = 3), S177C (72.4 ± 1.7%, n = 3), V178C (88.1 ± 3.2%, n = 4), V180C (65.2 ± 2.0%, n = 4), A181C (76.3 ± 2.0%, n = 4), D183C (46.0 ± 8.7%, n = 6) and R186C (44.8 ± 1.7%, n = 4). MTSEA-biotin potentiated IGABA at N188C (31.3 ± 10%, n = 3). An apparent lack of reaction (as in the case of A175C, V179C, E182C, and D191C) may indicate that no reaction has occurred or that the outcome of modification is functionally silent. It should be noted that most residues in this region were modified, although the magnitude of the effect of modification did not always achieve statistical significance (e.g. G184C, S185C, and Y190C). The pattern of solvent accessibility is not indicative of either a beta -strand or an alpha -helix, suggesting that this domain of the GABAAR alpha 1 subunit adopts either a loop or a random coil conformation (Fig. 6).

MTSEA-biotin Rates of Reaction-- The rate at which MTSEA-biotin reacts with a cysteine side chain depends mainly on the ionization of the thiol group and the access route to the engineered cysteine (18). A residue in a relatively open, aqueous environment will react faster than a residue in a relatively restrictive, non-polar environment. To gain insight into the physico-chemical environment of the loop F region of the GABA-binding site, we determined the reaction rate of MTSEA-biotin with several accessible cysteine mutants (Fig. 4). The rate MTSEA-biotin modified A181C was ~400-fold faster than the slowest reacting cysteine mutant, V180C. The rank order k2 values were A181C > R186C = R176C approx  S177C > D183C approx  V180C = V178C (Table II).


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Fig. 4.   Rate of MTSEA-biotin modification of D183C, V180C, and V178C. A, representative GABA-evoked (EC50) current traces following successive application (10-20 s) of MTSEA-biotin (50 µM) on alpha 1(D183C)beta 2 receptors in the absence and presence of SR-955531 (40 × KI) and GABA (5 × EC50). B, sequential application of MTSEA-biotin reduced the amplitude of subsequent GABA-mediated (EC50) currents. Data were normalized to the current measured at t = 0 for each experiment and plotted as a function of cumulative MTSEA-biotin exposure. Data were fit to a single exponential function to obtain a pseudo-first order rate constant (k). Second order rate constants (k2) were calculated by dividing the pseudo-first order rate constant by the concentration of MTSEA-biotin used (50 µM). Data points represent the mean ± S.E. for control (black-square), GABA (open circle ), SR95531() for at least three independent experiments. k2 values are shown in Table II.


                              
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Table II
Second order rate constants for MTSEA-mediated modification of accessible cysteine residues in the absence and presence of SR-95531, GABA, and pentobarbital
Data represent the mean ± S.E. of three to six independent experiments (n) carried out as described under "Experimental Procedures." Second order rate constants (k2) were calculated by dividing the pseudo-first order rate constant by the concentration of MTSEA-biotin used in the experiments. The concentrations of MTSEA-biotin used were 50 µM (R176C, V178C, V180C, D183C), 500 nM (A181C), or 5 µM (R186C). GABA (5 × EC50), SR-95531, (40 × KI), or pentobarbital (500 µM) was co-applied with MTSEA-biotin to determine their ability to alter the rate of covalent cysteine modification. (*, p < 0.05; **, p < 0.01, from control.)

Effects of GABA and SR-95531 on MTSEA-biotin Rate Constants-- To determine whether a given cysteine residue lines the neurotransmitter binding pocket, the rate of MTSEA-biotin modification of an introduced cysteine is measured in the presence of GABA and the competitive antagonist, SR-95531. We identify a residue as being within or near the binding site if the rate of covalent modification of the introduced cysteine is slowed in the presence of both agonists and antagonists, which presumably promote different conformational changes within the site. SR-95531 slowed the rate of modification at V178C, V180C, and D183C by factors of 3.6, 1.9, and 3.5, respectively (Fig. 4, Table II). GABA slowed the rate of reaction at R176C, V178C, V180C, and D183C (2.4-, 1.9-, 1.8-, and 3.5-fold, respectively). Protection of V178C, V180C, and D183C from covalent modification by MTSEA-biotin by GABA and SR-95531 suggests that the slowing of the MTSEA-biotin reaction rate results from steric block rather than allosteric changes induced in the protein. It is interesting to note that R176C was protected only by GABA but not SR-95531. S177C was protected significantly only by SR-95531. While the effects of GABA failed to reach statistical significance for this mutant, there was a clear trend in the data to suggest that GABA also slowed the MTSEA-biotin reaction rate (Table II, Fig. 5).


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Fig. 5.   Summary of the effects of GABA, SR-95531, and pentobarbital on MTSEA-biotin second order rate constants. Data were normalized to control second order rate constants (rate measured when no other compound was present). Co-application of GABA (5 × EC50) or SR95531 (40 × KI) slowed reaction of MTSEA-biotin at receptors containing the following mutations: R176C, V178C and D183C, suggesting that they line the GABA-binding site. Data represent the mean ± S.E. for at least three experiments. R176C was protected only by GABA, and while not significant, there is a clear trend in the data to suggest that S177C was protected by both agonist and antagonist. The rate of covalent modification at V180C, A181C, and R186C is significantly altered by pentobarbital (500 µM). *, p < 0.05.

Effects of Pentobarbital on MTSEA-biotin Rate Constants-- At wild-type alpha 1beta 2 or alpha 1beta 2gamma 2 GABAAR, the apparent affinity for direct activation by pentobarbital ranges from 500-700 µM (8, 10). Further, the mean single channel conductances elicited by GABA and pentobarbital are not different, suggesting that the open states produced by both ligands is similar (40). Moreover, mutations that compromise the affinity of GABA have thus far not affected the affinity or efficacy of barbiturates (8, 10), suggesting that the actions of pentobarbital are mediated from a site distinct from the GABA-binding site. Therefore, pentobarbital can be used as a pharmacological tool to assess gating-induced changes in the GABA-binding site. The rate of modification at R186C was slowed 3.2-fold in the presence of pentobarbital, while the rates of covalent modification at V180C and A181C were accelerated 1.4- and 2.3-fold, respectively (Table II, Fig. 5). Thus, these residues act as reporters of barbiturate-mediated channel gating.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Structure of the GABA Binding Pocket-- Previous work has shown that the GABA binding pocket is composed of aromatic (alpha 1Phe64, beta 2Tyr97, beta 2Tyr157, beta 2Tyr205), hydroxylated (beta 2Thr160, beta 2Thr202, beta 2Ser204, beta 2Ser209), and charged amino acid residues (alpha 1Arg66, beta 2Arg207). Here, our data demonstrating that GABA and SR-95531 protect V178C, V180C, and D183C also indicate that residues in loop F are near the agonist-binding site. An additional residue, Arg176, may be important for interactions with the agonist alone as modification of R176C was protected by GABA and not SR-95531. Barbiturate-mediated receptor activation did not alter MTSEA modification of R176C, suggesting that the observed slowing of the derivatization of R176C by GABA was a function of steric block, as opposed to channel-gating phenomena. Ligands of divergent chemical structure such as GABA and SR-95531 likely have different contact points within the GABA-binding site (3). However, the amino acid residues identified here need not be contact points for agonist/antagonist molecules, but they may be important for stabilizing the structure of the GABA-binding site or mediating local movements important for activation and/or desensitization.

When mapped onto a homology model of the GABA binding site, these residues appear to be located at the putative entrance of the binding site (Fig. 6). Using this model, we measured distances between loop F GABA-binding site residues and core GABA-binding regions. For example, approximate distances (alpha -beta , in Å) include the following: Asp183-Phe200 (9.0), Asp183-Thr202 (16.0), Asp183-Tyr205 (15.0), and Asp183-Arg207 (12.0); (alpha -alpha ,in Å) Asp183-Phe64 (12.0) Asp183-Arg66 (9.0). Whether these distances reflect the binding site in a resting, open, or desensitized state is unknown. The AChBP was crystallized in an ill-defined state, lacks an ion channel, and shows little cooperativity in ligand binding (39, 41). In addition, the loop F region was not well defined in the AChBP structure (17).


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Fig. 6.   A, model of the GABA-binding site at the beta -alpha subunit interface illustrating the random coil structure of the alpha 1 subunit loop F protein segment. Regions colored cyan correspond to cysteine mutants that were not accessible to MTSEA-biotin modification, and those residues that were accessible are colored yellow. B, GABA-binding site residues Val178 and Val180 (red) and Asp183 (blue) are illustrated. C, position of Asp183 in relation to other core GABA-binding site residues alpha 1Phe64 and alpha 1Arg66 from loop D (yellow), in addition to beta 2Arg207 and beta 2Tyr205 of loop C (red). Shown also is the predicted theoretical distance (12.0 Å) between beta 2Arg207 and alpha 1Asp183. The predicted distances between alpha 1Asp183 and other core binding site residues are summarized under "Discussion."

Previous work has demonstrated that the nAChR loop F is involved in agonist binding. Using a chemical cross-linker, Czajkowski and Karlin identified several negatively charged residues in loop F (delta Asp180, delta Glu182, and delta Glu189) within 9 Å of the Cys192/Cys193 loop of the alpha  subunit (13). These data suggest that, at least in some cases, the loop F domain of the delta  subunit is in close proximity to residues on the alpha  subunit that are within the core of the ACh-binding site. In addition, recent studies have shown that naturally occurring mutations in the loop F protein chain of the epsilon  subunit (D175N, N182Y) alter ACh microscopic binding affinity and channel gating (16).

Structural Rearrangements during Gating Transitions-- Allosteric proteins such as ligand-gated ion channels cycle through a number of affinity states, including a low affinity resting state, an active open channel state of moderate affinity and two desensitized states of high and very high affinity, respectively (42). During these state transitions, a molecule of GABA likely contacts a number of different residues. Residues important in the initial docking of the ligand may be different than residues involved in stabilizing ligand binding in open and desensitized states. It is likely that the GABA-binding site undergoes a series of transitions in which alternate domains of the protein are brought into closer contact with the ligand during active and desensitized states. It is equally possible that ligand interactions with amino acids in the inactive state are entirely different from those in the active and desensitized states (1), further complicating analysis of agonist binding segments.

Methanethiosulfonate reagents can be used as reporter molecules to detect agonist- or drug-induced changes in protein regions that are distant from the agonist or modulator binding site. GABA-induced structural rearrangements have been reported in the benzodiazepine-binding site (19) and in the alpha 1 subunit M2-M3 loop (43). The allosteric modulators, diazepam and propofol, induce changes in the alpha 1 subunit M3-spanning segment (35, 44). In addition, we have previously demonstrated movements within the GABA-binding site in response to pentobarbital gating of the channel (3, 10).

To test the hypothesis that movement of loop F is a plausible ion channel activation mechanism (14), we measured the rate of covalent modification of accessible amino acid residues in the presence of pentobarbital (500 µM). The ability of pentobarbital to alter the rates of modification of the loop F segment provides an indirect measure of changes that occur within this region of the binding cleft in the transition from the resting to the active/desensitized states. Co-application of pentobarbital and MTSEA-biotin should capture a receptor state that differs from that captured by application of MTSEA-biotin alone. Pentobarbital-mediated acceleration of the rate of modification at V180C (a GABA-binding site residue) and A181C and the concomitant slowing of the rate of modification of R186C indicate that Val180 and Ala181 move to a more accessible environment, while Arg186 becomes less accessible. These data demonstrate that the loop F region of the GABA binding site undergoes conformational rearrangements during receptor activation and/or desensitization. Other movements within the binding site may also be needed to trigger channel gating. For example, rotations and/or tilting movements of the beta 2 subunit may move the loop C region of the GABA-binding site closer to alpha 1 subunit binding segments (45).

    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

SCAM analysis has enabled us to identify novel residues of the alpha 1 subunit (Val178, Val180, and Asp183) that contribute to forming the GABA-binding site. Further, we provide evidence that the domain defined by Pro174-Asp191 adopts a random coil/turn conformation. Barbiturate-mediated channel activation suggests that this segment of the protein undergoes conformational movements during channel gating. We speculate that this loop of the protein is a dynamic element that may move closer to the core of the binding site during allosteric transitions to higher affinity states. While this is a plausible channel-gating mechanism, corroboration of these SCAM observations will require studies using chemical cross-linkers to understand the relative positions of amino acids in this domain during the transduction of agonist binding to channel opening and desensitization.

    ACKNOWLEDGEMENTS

We thank James Seffinga-Clark for his expertise in the preparation of Xenopus oocytes and in homology modeling and Dr. Mary Lindstrom for her expert statistical analysis. We thank Amy M. Kucken for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by NINDS, National Institutes of Health Grant 34727 (to C. C.) and a postdoctoral fellowship (to J. G. N.) from the Natural Sciences and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Physiology, Rm. 197 MSC, University of Wisconsin-Madison, 1300 University Ave., Madison, WI 53706. Tel.: 608-265-5863; Fax: 608-265-5512; E-mail: czajkowski@physiology.wisc.edu.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M211905200

    ABBREVIATIONS

The abbreviations used are: GABAAR, gamma -aminobutyric acid type A receptor; MTSEA, N-biotinylaminoethyl methanethiosulfonate; SCAM, substituted-cysteine accessibility method; nAChR, nicotinic acetylcholine receptor; AChBP, acetylcholine-binding protein.

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CONCLUSIONS
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