Mutation of Glutamate 155 of the GABA_A Receptor β₂ Subunit Produces a Spontaneously Open Channel: A Trigger for Channel Activation

Abstract

Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA_A receptor (GABA_AR) ligand-binding domain predicts that β₂Glu¹⁵⁵ is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the β₂Ile¹⁵⁴-Asp¹⁶³ region in GABA_AR activation, each residue was individually mutated to cysteine and coexpressed with wild-type α₁ subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC₅₀ value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) K_I value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that β₂Thr¹⁶⁰ and β₂Asp¹⁶³ are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of β₂Glu¹⁵⁵ resulted in spontaneously open GABA_ARs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto β₂E155C closed the spontaneously open channels, suggesting that β₂Glu¹⁵⁵ is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the β₂ Ile¹⁵⁴-Asp¹⁶³ region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.

Introduction

Agonists and antagonists induce different molecular rearrangements in neurotransmitter-binding sites of ligand-gated ion channels (LGICs) (Armstrong and Gouaux, 2000; Boileau et al., 2002; Chang and Weiss, 2002). Agonists, but not antagonists, promote opening of the ion channel pore. It is likely that movements of amino acids near or within the neurotransmitter recognition site trigger the cascade of events leading to channel opening (Boileau et al., 2002; Torres and Weiss, 2002; Unwin et al., 2002; Miyazawa et al., 2003; Chakrapani et al., 2004). Here, we examined the I1e¹⁵⁴-Asp¹⁶³ region of the GABA_A receptor (GABA_AR) β₂ subunit to identify residues that mediate local movements within the binding site that initiate channel gating and residues involved in GABA binding.

GABA_ARs are heteropentameric LGICs that mediate fast synaptic inhibitory neurotransmission in the brain. The α₁β₂γ₂ GABA_AR subtype is the most abundant in vivo, and heterologous expression studies suggest a β-α-β-α-γ stoichiometry and subunit arrangement (Chang et al., 1996; Tretter et al., 1997; Farrar et al., 1999; Baumann et al., 2001, 2002). Expression of α and β subunits also gives rise to functional GABA_AR with putative stoichiometries of either 3α:2β (Im et al., 1995) or 3β:2α (Baumann et al., 2001; Horenstein et al., 2001) that lack sensitivity to benzodiazepines (Schofield et al., 1987; Pritchett et al., 1989), are responsive to barbiturates, and have a high apparent affinity for agonists (Boileau et al., 1999, 2002; Wagner and Czajkowski, 2001).

A recent homology model of the GABA_AR agonist-binding site predicts that β₂Glu¹⁵⁵ interacts with the positively charged moiety of GABA (Cromer et al., 2002). In addition, mutagenesis studies have determined that nearby residues, β₂Tyr¹⁵⁷ and β₂Thr¹⁶⁰, are important for GABA binding (Amin and Weiss, 1993). Similarly, amino acid residues in aligned regions of the muscle-type nicotinic acetylcholine α1 (Trp¹⁴⁸, Tyr¹⁵¹, and Asp¹⁵²) (Dennis et al., 1988; Galzi et al., 1991; Sugiyama et al., 1996), glycine α1 (Asp¹⁴⁸, Gly¹⁶⁰, and Tyr¹⁶¹) (Vandenberg et al., 1992, 1993), and serotonin type 3 receptor subunits (Trp¹⁶⁰) (Spier and Lummis, 2000) have been determined to be critical for agonist-antagonist binding (see Fig. 1) and define region “B”^a of the ligand-binding site. The contributions of these residues in forming their respective agonist binding sites are supported by homology models that place this region (from β-strand 7 and loop 8) within the putative core of LGIC neurotransmitter-binding sites (Cromer et al., 2002; Holden and Czajkowski, 2002; LeNovère et al., 2002; Newell and Czajkowksi, 2003; Reeves et al., 2003).

Figure 1.

The Ile¹⁵⁴-Asp¹⁶³ segment (region B) of the rat GABA_AR β₂ subunit is aligned with homologous segments of the Torpedo nAChR α, glycine receptor (GlyR) α₁, and 5-HT_3a subunits. The numbering reflects the position of the residues in the mature GABA_AR β₂ subunit. Circled residues β₂Tyr¹⁵⁷ and β₂Thr¹⁶⁰ are implicated in GABA binding (Amin and Weiss, 1993). Residues important for ligand recognition in other binding sites are boxed. nAChR αTrp¹⁴⁹ and possibly Tyr¹⁵¹ and Asp¹⁵² have been implicated in forming the acetylcholine-binding site (Dennis et al., 1988; Galzi et al., 1991; Sugiyama et al., 1996). GlyR α₁Gly¹⁶⁰, Tyr¹⁶¹ (Vandenberg et al., 1992; Schmieden et al., 1993), and Phe¹⁵⁹ (Schmieden et al., 1993) are important for antagonist recognition, whereas 5-HT_3a Trp¹⁶⁰ (Spier and Lummis, 2000) is important for the actions of serotonin. nAChR αG158S has been reported as a naturally occurring mutation in myasthenia gravis patients that gives rise to “slow channels” (Sine et al., 1995; Croxen et al., 1997) GABA_AR β₂ subunit residues accessible to MTSEA-biotin modification are underlined (E155C, G158C, T160C, and D163C).

Here, we demonstrate that expression of α₁β₂(E155C) gives rise to spontaneously open GABA channels. Mutation of β₂Glu¹⁵⁵ alters both channel-gating properties and impairs agonist binding. In addition, we provide evidence that β₂Thr¹⁶⁰ and β₂Asp¹⁶³ are found on an aqueous surface within or near the GABA-binding site and undergo conformational rearrangements during pentobarbital-mediated gating events. Together, the data suggest that movement in this region of the GABA-binding site is one of the initial triggers for coupling binding to gating.

Materials and Methods

Mutagenesis and expression in oocytes. Rat cDNAs encoding the GABA_AR α₁ and β₂ subunits were used in this study. The β₂ cysteine mutants were engineered using a recombinant PCR method, as described previously (Boileau et al., 1999; Kucken et al., 2000). Cysteine substitutions were made in the β₂ subunit at Ile¹⁵⁴, Glu¹⁵⁵, Ser¹⁵⁶, Tyr¹⁵⁷, Gly¹⁵⁸, Tyr¹⁵⁹, Thr¹⁶⁰, Thr¹⁶¹, Asp¹⁶², and Asp¹⁶³ (see Fig. 1). Cysteine substitutions were verified by restriction endonuclease digestion and double-stranded DNA sequencing.

All wild-type and mutant cDNAs were subcloned into the vector pGH19 (Liman et al., 1992; Robertson et al., 1996) for expression in Xenopus laevis oocytes. Oocytes were isolated as described previously (Boileau et al., 1998). cRNA transcripts were prepared using the T7 mMessage machine (Ambion, Austin, TX). GABA_A receptor α₁ and β₂ or β₂ mutant subunits were coexpressed by injection of cRNA (675 pg/subunit) in a 1:1 ratio (α:β). The oocytes were maintained in modified ND96 medium [containing (in mm): 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, and 5 HEPES, pH 7.4] that had been supplemented with 100 μg/ml gentamicin and 100 μg/ml bovine serum albumin. Oocytes were used 2-7 d after injection for electrophysiological recordings.

Two-electrode voltage-clamp analysis. Oocytes under two-electrode voltage clamp were perfused continuously with ND96 recording solution [containing (in mm): 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, 5 HEPES, pH 7.4] 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 performed using a GeneClamp500 Amplifier (Axon Instruments, Foster City, CA). Borosilicate electrodes were filled with 3 m KCl and had resistances of 0.5-3.0 MΩ in ND96. Stock solutions of GABA, 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) and piperidine-4-sulfonic acid (P4S) (Sigma, St. Louis, MO) were prepared in water, whereas stock solutions of picrotoxin (PTX) (Sigma) and N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin; 100 mm; Biotium, Hayward, CA) were prepared in dimethylsulfoxide (DMSO). All compounds were diluted appropriately in ND96 such that the final concentration of DMSO was ≤2%. The vehicle did not affect GABA-activated currents.

To measure the sensitivity to GABA or P4S, the agonist (0.0001-100 mm) or partial agonist (0.00001-10 mm) was applied via gravity perfusion or by pipette application (∼5-8 sec) with a 3-15 min washout period between each application to ensure complete recovery from desensitization. Peak GABA- or P4S-activated current (I_GABA or I_P4S) was recorded. To correct for slow drift in the amplitude of the response as a function of time, concentration-response data were normalized to a low concentration of agonist (EC₂-EC₅). The apparent affinity of pentobarbital using concentrations between 0.01 and 10 mm was determined via gravity perfusion (∼5-8 sec) with a 3-5 min washout period between each application. Peak pentobarbital-activated current was recorded. Concentration-response data for pentobarbital were normalized to a previous application of pentobarbital (100 μm). Concentration-response curves for GABA, P4S, or pentobarbital were generated for each recombinant receptor, and the data were fitted by nonlinear regression analysis using Prism software (GraphPad, San Diego, CA). Data were fitted to the following equation: I = I_max/(1 + (EC₅₀/[A])ⁿ), where I is the peak amplitude of the current for a given concentration of GABA, P4S, or pentobarbital ([A]), I_max is the maximum current, EC₅₀ is the concentration required for half-maximal receptor activation, and n is the Hill coefficient.

To determine SR-95531 or PTX IC₅₀ values, GABA (EC₅₀) was applied via gravity perfusion followed by a brief washout period (20 sec) before application of GABA (EC₅₀) and increasing concentrations of SR-95531 or PTX. The response to the application of SR-95531/PTX and GABA was normalized to the response elicited by the agonist alone. Concentration-inhibition curves were generated by nonlinear regression analysis using GraphPad Prism software. Data were fitted 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. SR-95531 K_I values were calculated using the Cheng-Prussof correction: K_I = 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 (Cheng and Prussof, 1973).

Modification of cysteine residues by MTSEA-biotin. MTSEA-biotin was the cysteine-specific reagent used in this study because it is a relatively impermeant compound (Daniels and Amara, 1998), the dimensions (14.5 Å unreacted moiety; 11.2 Å reacted moiety) of which are similar to SR-95531 (13.5 Å) but longer than GABA (4.5 Å). Therefore, it is reasonable to assume that MTSEA-biotin can occupy the GABA-binding site and that this reagent will principally modify extracellular cysteine residues. We used the following criterion for stability of the response for these studies: ≤10% variation in I_GABA (EC₅₀) on two consecutive applications at regular intervals (10 min). Oocytes were then allowed to recover fully, after which a high concentration of MTSEA-biotin (2 mm) was applied (2 min). After MTSEA-biotin application, cells were washed (5 min) with ND96, after which GABA (EC₅₀) was reapplied to determine the effect of MTSEA-biotin application on I_GABA. Effects of MTSEA-biotin were calculated as the difference in the amplitude of the GABA-gated current before and after MTSEA-biotin application as follows: (I_GABAPRE - I_GABAPOST/I_GABAPRE) × 100, where “post” refers to the amplitude of the GABA-gated current after MTSEA-biotin application, and “pre” refers to the amplitude of the stabilized GABA-gated current before covalent modification by MTSEA-biotin.

Rate of MTSEA-biotin reaction. The rate at which MTSEA-biotin modified introduced cysteine residues (E155C, T160C, and D163C) was measured using low MTSEA-biotin concentrations, as described previously (Newell and Czajkowski, 2003). The concentrations of MTSEA-biotin used were 50 nm (D163C), 100 nm (T160C), and 2 mm (E155C). The experimental protocol is described as follows: GABA (EC₅₀) application (5 sec), ND96 washout (25 sec), MTSEA-biotin application (10-20 sec), ND96 washout (2.2-2.3 min). The sequence was repeated until I_GABA no longer changed after the MTSEA-biotin treatment (i.e., the 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 coapplying GABA (5 × EC₅₀), SR-95531 (6 × K_I), or pentobarbital (50 or 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) after exposure to high concentrations of GABA, SR-95531, or pentobarbital were stable. This ensured (1) a complete washout of drugs and that (2) reductions in the current amplitude resulted from the application of MTSEA-biotin.

For all rate experiments, the decrease in I_GABA 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 (Pascual and Karlin, 1998). Second-order rate constants were determined using at least two different concentrations of MTSEA-biotin to ensure accuracy of the protocol.

Statistical analysis. Log (EC₅₀) values, log (K_I) values, and log (k₂) rates were analyzed using a one-way ANOVA followed by a Dunnett's post hoc test to determine levels of significance.

Structural modeling. The mature protein sequences of the rat α₁ and β₂ subunits were homology modeled with a subunit of the ACh binding protein (AChBP) (Brejc et al., 2001). The crystal structure of the AChBP was downloaded from RCSB Protein Data Bank (code 1I9B) and loaded into Swiss Protein Bank Viewer (SPDBV). The α1 protein sequence from Thr¹²-Ile²²⁷ and the β₂ protein sequence from Ser¹⁰-Leu²¹⁸ were aligned with the AChBP primary amino acid sequence as depicted by Cromer et al. (2002) and threaded onto the AChBP tertiary structure using the “Interactive Magic Fit” function of SPDBV. The threaded subunits were imported into SYBYL (Tripos, St. Louis, MO), in which energy minimization was performed (<0.5 kcal/Å). The first 100 iterations were performed using Simplex minimization (Press et al., 1988) followed by 1000 iterations using the Powell conjugate gradient method (Powell, 1977). A β₂/α₁ GABA-binding site interface was assembled by overlaying the monomeric subunits on the AChBP scaffold, and the resulting structure was imported into SYBYL and energy minimized. Neither water nor entropy factors were included during the minimizations. After the global energy minimization, several TRIPOS programs were run to evaluate the accuracy of the model. Ramachandran plots, χ plots, side-chain positions, and cis- and trans-bonds were all examined. Problems in the structure that were revealed by these evaluations were fixed manually, and energy minimizations were run again as needed. Our model is quite similar to models published recently for the nicotinic ACh receptor (nAChR) and GABA_AR ligand-binding domains (Cromer et al., 2002; LeNovère et al., 2002). Regions with insertions were modeled by fitting structures from a loop database. Because the sequence identity of the AChBP and the GABA_AR extracellular ligand-binding domain is only 18%, caution must be used in interpreting the absolute positions of individual side-chain residues in the model.

Results

Expression and functional characterization of cysteine mutants

Cysteine substitutions were engineered at 10 individual positions (Fig. 1) in the GABA_AR β₂ subunit (Ile¹⁵⁴, Glu¹⁵⁵, Ser¹⁵⁶, Tyr¹⁵⁷, Gly¹⁵⁸, Tyr¹⁵⁹, Thr¹⁶⁰, Thr¹⁶¹, Asp¹⁶², and Asp¹⁶³), coexpressed with wild-type α₁ subunits in X. laevis oocytes, and analyzed using two-electrode voltage clamp. Expression of most mutant β₂ subunits produced functional channels (I_GABA = 1-10 μA), with the exceptions Y157C and Y159C. We speculate that introduction of cysteine residues at these positions impaired assembly of mutant receptors.

For the cysteine mutants that did express, seven of eight significantly increased GABA EC₅₀ values, demonstrating that this region is particularly sensitive to structural perturbation. Expression of receptors containing I154C, E155C, S156C, G158C, T160C, D162C, and D163C increased GABA EC₅₀ values 8-, 3375-, 22-, 260-, 23-, 18-, and 9-fold relative to wild type (1.6 μm) (Fig. 2A, Table 1). The Hill coefficients for GABA activation of G158C- and D163C-containing receptors were significantly lower than wild type. The K_I values for the competitive antagonist SR-95531 were significantly different from wild type (K_I = 163 nm) for E155C, S156C, and G158C by 11-, 3-, and 18-fold, respectively (Table 1, Fig. 2B). Small currents (I_max < 90 nA) of receptors containing G158C precluded additional analysis.

Figure 2.

A, Concentration-response curves of GABA-activated current for wild type (▪), E155C (▴), G158C (○), T160C (♦), and D163C (□) expressed in Xenopus oocytes. Data were normalized to peak I_GABA for each experiment. Data points represent the mean ± SE of three to five independent experiments. B, Concentration dependence of SR-95531 inhibition of I_GABA (EC₅₀) current for wild type (▪), G158C (○), and E155C (▴). Data points represent the mean ± SE of three independent experiments. The GABA EC₅₀ values, SR-95531 K_I values, and calculated Hill coefficients are reported in Table 1.

Table 1.

Concentration-response data for GABA activation and SR-95531 inhibition of wild-type and mutant receptors expressed in Xenopus oocytes

	GABA							SR-95531
Receptor	EC50 (μm)	nH	n	mut/wt	KI (nm)	nH	n	mut/wt
α1β2	1.6 ± 0.2	1.35 ± 0.15	5	1	163 ± 83	1.13 ± 0.10	3	1
α1β2(I154C)	12.1 ± 4.7**	1.13 ± 0.22	3	7.6	143 ± 63	0.87 ± 0.10	3	0.9
α1β2(E155C)	5400 ± 1500**	1.21 ± 0.07	3	3375	1810 ± 401*	0.66 ± 0.10	3	11.1
α1β2(S156C)	34.5 ± 8.8**	1.32 ± 0.15	3	22	561 ± 178*	0.76 ± 0.15	3	3.4
α1β2(Y157C)	No current
α1β2(G158C)	416 ± 184**	0.65 ± 0.09**	3	260	2920 ± 1100*	1.35 ± 0.40	3	17.9
α1β2(Y159C)	No current
α1β2(T160C)	37.4 ± 11.7**	1.40 ± 0.10	6	23	250 ± 88	1.03 ± 0.09	3	1.5
α1β2(T161C)	1.8 ± 0.4	1.10 ± 0.14	3	1.1	132 ± 38	1.03 ± 0.06	3	0.8
α1β2(D162C)	28.5 ± 5.6**	1.48 ± 0.13	4	18	187 ± 115	1.49 ± 0.7	3	1.1
α1β2(D163C)	13.8 ± 3.8*	0.82 ± 0.05*	3	8.6	120 ± 32	0.84 ± 0.12	3	0.7

Data represent the mean ± SE for three to six experiments (n). EC₅₀ values, K_I values, and Hill slopes (n_H) were determined from concentration-response data using nonlinear regression analysis with GraphPad Prism software. Hill slopes, log(EC₅₀), and log(K_I values were analyzed using a one-way ANOVA followed by a Dunnett's test to determine the levels of significance (^*p < 0.05; ^**p < 0.01; from wild type). mut, Mutant; wt, wild type.

Pentobarbital is a barbiturate that exerts its pharmacological effects (allosteric modulation and channel opening) via interactions with the GABA_A receptor at a binding site distinct from the GABA site (Akk and Steinbach, 2000). Pentobarbital is therefore useful for assessing the consequences of mutating residues located near the GABA-binding site on overall receptor structure-function. Pentobarbital activated wild-type receptors with an EC₅₀ of 1.1 ± 0.3 mm (n = 4) (Table 2) but failed to elicit current in receptors containing Y157C or Y159C, again suggesting that these mutant β₂ subunits did not assemble into functional channels. Sensitivity to pentobarbital was increased approximately twofold for E155C- and T160C-containing receptors [EC₅₀ values = 0.53 ± 0.1 mm (n = 4) and 0.32 ± 0.03 mm (n = 3), respectively], whereas expression of D163C (EC₅₀ = 2.0 ± 0.3 mm; n = 3) had no significant effect on pentobarbital EC₅₀. These data suggest that the rightward GABA EC₅₀ shifts (Table 1) measured for E155C-, T160C-, and D163C-containing receptors are attributable to local effects at the GABA-binding site.

Table 2.

Concentration-response data for GABA, P4S, SR-95531, PTX, and pentobarbital for α₁β₂ and α₁β₂(E155C) receptors expressed in Xenopus oocytes

	α1β2			α1β2(E155C)
Ligand	EC50/IC50(μm)	nH	EC50/IC50 (μm)	nH	mut/wt
GABAa	1.6 ± 0.2	1.35 ± 0.15	5400 ± 1500*	1.21 ± 0.07	3375
P4S	4.7 ± 0.4	1.59 ± 0.22	730.7 ± 52.1*	1.30 ± 0.11	152
SR-95531a	0.2 ± 0.08	1.13 ± 0.10	1.8 ± 0.4*	0.66 ± 0.1	11.1
PTX	4.7 ± 1.2	0.84 ± 0.02	3.1 ± 0.1	0.81 ± 0.10	0.6
Pentobarbital	1100 ± 300	1.56 ± 0.07	530 ± 100*	1.08 ± 0.02	0.5

Data represent the mean ± SE for more than three experiments. EC₅₀ values, IC₅₀ values, K_I values, and Hill slopes (n_H) were determined from concentration-response data using nonlinear regression analysis with GraphPad Prism software. Hill slopes, log(EC₅₀), log(IC₅₀), and log (K_I) values were analyzed using a two-tailed unpaired t test to determine the levels of significance (^*p < 0.01; from α₁β₂). mut, Mutant, wt, wild type.

^a Values are from Table 1.

Although it is impossible to know whether the introduced cysteine residues occupy positions equivalent to wild-type residues, because SR-95531 and pentobarbital apparent affinities were similar or, in certain cases, more potent for some mutant receptors in which there were large rightward shifts in GABA EC₅₀, we believe gross structural reorganizations of the GABA_A receptor did not occur as a result of these mutations.

Spontaneous openings at α₁β₂(E155C)

Expression of α₁β₂(E155C) receptors gave rise to higher than normal resting conductances (I_leak), the magnitudes of which (-609 ± 76 nA; n = 9) were ∼12-fold greater than injection-matched wild-type receptors (-51 ± 9 nA; n = 9). To determine the nature of this high resting conductance, we applied the GABA_AR channel blocker PTX. PTX (100 μm) reduced I_leak by 72.1 ± 4.2% (n = 3) (Fig. 3A), demonstrating that spontaneously open GABA_AR channels accounted for the high resting conductance. PTX inhibited GABA-activated currents elicited from α₁β₂(E155C) receptors with an IC₅₀ value of 3.1 μm, which was not significantly different from wild-type receptor values (4.7 μm) (Fig. 3B-D, Table 2).

Figure 3.

A, Representative current trace demonstrating that the high resting leak conductance for α₁β₂(E155C) receptors expressed in Xenopus oocytes is sensitive to blockade by PTX (100 μm). PTX (100 μm) reduced the resting conductance (-609 ± 76 nA; n = 9) by 72.1 ± 4.2%. Representative current traces obtained from PTX-mediated inhibition of GABA-evoked currents (EC₅₀) for wild-type (WT; B) and α₁β₂(E155C) (C) receptors are shown. Note that the PTX IC₅₀ value for the E155C mutant was determined by using the baseline leak current as the zero. D, Concentration dependence of PTX-mediated reduction of I_GABA (EC₅₀) current for wild type (▪) and E155C (○) expressed in Xenopus oocytes. Data points represent the mean ± SE of three independent experiments. Data were normalized to I_GABA in the absence of PTX. IC₅₀ values are summarized in Table 2.

Spontaneously active LGICs often arise as a consequence of mutations in the M2 channel-lining segment, and a characteristic of these constitutively open channels is a leftward shift in agonist concentration responses (Bertrand et al., 1992; Filatov and White, 1995; Labarca et al., 1995; Tierney et al., 1996; Chang and Weiss, 1998, 1999; Thompson et al., 1999; Findlay et al., 2000). However, this was not the case on expression of β₂E155C, wherein we observed a 3375-fold decrease in GABA sensitivity. The sensitivity of the partial agonist (P4S) was also reduced (with no apparent reduction in efficacy) (Fig. 4A,B), albeit to a lesser extent than that of GABA (152-fold) (Table 2, Fig. 4C). The mutation also decreased the sensitivity of the competitive antagonist SR-95531 (11.1-fold rightward shift) (Table 2). Again, it should be noted that for this mutation, pentobarbital sensitivity was increased twofold relative to wild type (Table 2, Fig. 4D). Mutation of ρ₁Y102 located in the GABA_C receptor D region of the agonist binding site (i.e., β-strand 2) has also been reported to result in spontaneously open channels with similar properties (Torres and Weiss, 2002).

Figure 4.

Representative current traces obtained from GABA- and P4S-mediated activation of wild-type (WT; A) and α₁β₂(E155C) (B) receptors are shown. P4S efficacy at wild-type (0.41 ± 0.05; n = 7) and α₁β₂(E155C) (0.47 ± 0.04; n = 6) receptors was not significantly different, where efficacy is reported as I_{max P4S}/I_{max GABA.} Values are given as mean ± SE. C, Concentration-response curves for P4S-activated current for wild-type (▪) and α₁β₂(E155C) (○) receptors expressed in Xenopus oocytes. D, Concentration-response curves for pentobarbital-activated current for wild-type (▪) and α₁β₂(E155C) (○) receptors. Data points represent the mean ± SE of three to four independent experiments. The EC₅₀ values for P4S and pentobarbital are reported in Table 2.

Alterations in EC₅₀ values are difficult to evaluate because changes in either ligand binding and/or channel gating can alter this macroscopic constant (Colquhoun, 1998). The increase in open probability for the E155C mutant indicates that the mutation altered GABA_A receptor channel gating. If the mutation altered gating exclusively, similar-fold changes in the apparent affinities of a series of ligands as well as leftward shifts in their concentration responses would be expected (Zhang et al., 1994). The apparent affinities for GABA, P4S, and SR-95531 were altered by different factors (Table 2), and rightward shifts in their concentration responses were observed. Thus, these data indicate that, besides altering gating, E155C decreased the binding of orthosteric ligands to the GABA-binding site. We can exclude the possibility that these differential effects arise from a mixed population of receptors [i.e.,α₁β₂(E155C) and β₂(E155C)] because expression of β₂E155C alone produced no currents.

In addition to PTX, covalent modification of E155C by thiol-specific reagents closed the spontaneously open channels. The leak current was reduced by MTSEA-biotin (2 mm; 57.2 ± 0.7%; n = 3), MTSEA-biotin-CAP (N-biotinylcaproylaminoethyl methanethiosulfonate) (2 mm; 63.0 ± 1.8%; n = 3), 2-aminoethyl methanethiosulfonate (MTSEA) (2 mm; 39.4 ± 1.4%; n = 3), MTSET (2-(trimethylammonium)ethyl methanethiosulfonate) (2 mm; 30.5 ± 0.9%; n = 3), and MTSES (2-sulfonatoethyl methanethiosulfonate) (2 mm; 26.8 ± 7.5%; n = 3) (Fig. 5) The observation that I_leak is reduced by tethering different chemical groups directly onto E155C suggests that this region of the binding site may play a key role in the triggering of allosteric transitions from the closed to open state.

Figure 5.

Representative traces demonstrating the effects of PTX (100 μm; A) and MTSEA (2 mm; B) on I_leak for α₁β₂(E155C) receptors are shown. Reagents were applied in the absence of GABA and resulted in a reduction in the leak current. Note that the PTX trace is the same as that presented in Figure 3A but is included here for comparison purposes. C, Bar graph summarizing the effects of all methanethiosulfonate reagents on I_leak. Note that the maximum effect of each MTS reagent is normalized to the maximum effect obtained with 100 μm PTX for comparison purposes. The mean values for blockade of the resting conductance by MTS reagents are reported in Results. Error bars represent SE.

MTSEA-biotin modification of cysteine residues

To further examine the β₂ subunit Ile¹⁵⁴-Asp¹⁶³ region, we assessed the accessibility of cysteine residues introduced into this region. Wild-type and mutant receptors were exposed to MTSEA-biotin (2 mm; 2 min), a thiol-specific reagent that modifies water-accessible cysteine residues (Karlin and Akabas, 1998). MTSEA-biotin significantly reduced I_GABA for receptors containing E155C (91.8 ± 1.5%; n = 6), G158C (34.6 ± 4.5%; n = 4), T160C (60.9 ± 3.0%; n = 10), and D163C (98.9 ± 3.7%; n = 8) (Fig. 6). MTSEA-biotin had no effect on wild-type receptors or those containing I154C, S156C, T161C, and D162C. Lack of effect indicates that the thiol group was not accessible to modification or that modification produced no detectable functional effect. The accessibility pattern of the residues is consistent with the predicted side-chain positions observed in homology models of the GABA_A receptor, which envision this region of the GABA_A receptor forming a loop structure (Cromer et al., 2002).

Figure 6.

Summary of the effects of MTSEA-biotin (2 mm) on wild-type (WT) and mutant receptors. A, Representative current traces demonstrating the effects of MTSEA-biotin application on GABA-mediated current (EC₅₀) at wild-type and T160C- and D163C-containing receptors. The arrows in the current traces represent MTSEA-biotin application (2 min), and the breaks represent the subsequent wash (5 min). B, Summary of the effect of MTSEA-biotin at all receptors. The effect (percentage of inhibition or potentiation) is calculated using the following: ([I_{GABA-POST MTSEA-biotin}/I_{GABA-PRE MTSEA-biotin}] - 1) × 100. Bars represent the mean ± SE for 3-10 experiments. The filled bars indicate values that were statistically different from wild-type values (^*p < 0.001). Note that expression of Y157C or Y159C failed to yield functional receptors.

MTSEA-biotin rates of reaction

The rate of reaction of MTSEA-biotin with an introduced cysteine mainly depends on the ionization of the thiol group and the access route of the methanethiosulfonate reagent (Karlin and Akabas, 1998). Cysteine residues with ionized sulfhydryls react 10⁸ to 10⁹ times faster than nonionized sulfhydryls (Roberts et al., 1986). Second-order rate constants therefore provide information about the local environment of a substituted cysteine. The fast second-order rate constants (in the absence of other ligands) for MTSEA-biotin modification of D163C (604,771 m^-1sec^-1) and T160C (286,100 m^-1sec^-1) indicate that both residues are found in an open, aqueous environment. The second-order rate constant for E155C is significantly slower (27.9 m^-1sec^-1), suggesting that the thiol group is not well ionized and/or that the introduced cysteine residue is in a restricted-buried environment (Table 3, Fig. 7).

Table 3.

Second-order rate constants for MTSEA-biotin-mediated modification of accessible engineered cysteine residues in the absence (control) and presence of SR-95531, GABA, and pentobarbital

	Control			SR-95531			GABA			Pentobarbital(500)			Pentobarbital(50)
Receptor	k2 (m−1sec−1)	n	k2 (m−1sec−1)	n	k2 (m−1sec−1)	n	k2 (m−1sec−1)	n	k2 (m−1sec−1)	n
α1β2(E155C)	27.9 ± 11	3	24.8 ± 3.8	3	23.7 ± 3.7	3	27.5 ± 9.8	3	N.D.
α1β2(T160C)	288, 664 ± 24, 911	11	132, 200 ± 3, 524*	4	122, 828 ± 37, 753*	4	131, 768 ± 16, 610*	4	107, 493 ± 17, 751*	3
α1β2(D163C)	604, 771 ± 22, 681	7	293, 200 ± 43, 267*	3	280, 018 ± 43, 416*	3	281, 760 ± 56, 625*	3	479, 100 ± 48, 932	4

Data represent the mean ± SE of 3-11 independent experiments (n). k₂ values 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 2 mm (E155C), 100 nm (T160C), and 50 nm (D163C). GABA (5 × EC₅₀), SR-95531 (6 × K_I), and pentobarbital (500 and 50 μm) were coapplied as described in Materials and Methods to determine their ability to alter the rate of covalent cysteine modification. N.D., Not determined. ^*p < 0.01; from control.

Figure 7.

Rate of MTSEA-biotin modification of T160C and D163C. A, Representative GABA-evoked (EC₅₀) current traces after successive application (10-20 sec) of MTSEA-biotin on α₁β₂(D163C) receptors in the absence or presence of SR-95531 (6 × K_I) and GABA (5 × EC₅₀). Sequential application of MTSEA-biotin reduced the amplitude of subsequent GABA-mediated (EC₅₀) currents at receptors containing T160C (B) and D163C (C). 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 fitted to a single exponential function to get k₁. k₂ values were calculated by dividing the pseudo-first-order rate constant by the concentration of MTSEA-biotin used. Data points represent the mean ± SE for control (▪), GABA (□), SR-95531 (○), and pentobarbital (500 μm; •) for at least three independent experiments. Data are summarized in Table 3.

To determine whether a given residue lies near the neurotransmitter binding site, MTSEA-biotin reaction rates were measured in the presence of GABA and the competitive antagonist SR-95531. These ligands promote different conformational changes in the binding site (Boileau et al., 2002), and thus, if the rate at which MTSEA-biotin reacts with an introduced cysteine is slowed by both ligands, then it is likely that the ligands are sterically blocking the reaction and that the sulfhydryl side chain is facing into or near the GABA-binding site. Both GABA (at EC₉₀ concentration) and SR-95531 (at IC₉₀ concentration) significantly slowed modification of T160C and D163C approximately twofold (Fig. 8), suggesting that these residues are found within or near the GABA-binding site (Fig. 9). Although the data are consistent with GABA and SR-95531 causing a steric block, it is feasible that the binding of either ligand induces local allosteric changes in the receptor that leads to the slowing of MTSEA-biotin reaction rates. Neither ligand slowed modification of E155C (Table 3). Because α₁E155C receptors are spontaneously open, the control rate of MTSEA-biotin modification of E155C likely reflects reaction to a “ligand-bound, active” conformation of the binding site. Thus, the result that GABA and SR-95531 had no effect on modification rate is not surprising.

Figure 8.

Summary of the effects of GABA, SR-95531, and pentobarbital on MTSEA-biotin second-order rate constants. Data were normalized to the control k₂ (rate measured when no other compound was present; denoted as a dashed line). Coapplication of GABA (5 x EC₅₀; black), SR-95531 (6 x K_I; gray), and pentobarbital [500 μm (white) or 50 μm (lined)] slows reaction of MTSEA-biotin at receptors carrying T160C and D163C (^*p < 0.001; from control). Note that 50 μm pentobarbital significantly slows the rate of reaction at T160C but not at D163C. The ability of 50 μm pentobarbital to alter the rate of reaction at E155C was not determined (N.D.). Error bars represent SE.

Figure 9.

Top, Side view of a homology model of the GABA-binding site β (cyan)-α (yellow) subunit interface with residues β₂Glu¹⁵⁵, β₂Thr¹⁶⁰, β₂Asp¹⁶³ in region B and residue α₁Phe⁶⁴ in region D displayed as sticks. Middle, Position of β₂Glu¹⁵⁵ (region B) relative to β₂Arg²⁰⁷ (region C) is highlighted. β₂Arg²⁰⁷ has been identified previously as a GABA-binding site residue (Wagner and Czajkowski, 2001; Wagner et al., 2004). Bottom, View of the binding site from below, which shows the close apposition of β₂Glu¹⁵⁵ and β₂Arg²⁰⁷ (2.5 Å), suggesting an electrostatic interaction between the two residues.

Effect of pentobarbital on MTSEA-biotin second-order rate constants

To identify whether movements occur in and near the Ile¹⁵⁴-Asp¹⁶³ region of the GABA-binding site during channel gating and modulation, we measured the rates of reaction of MTSEA-biotin with T160C, D163C, and E155C in the presence pentobarbital. The ability of pentobarbital to alter the rates of modification 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. As a result of the slowness of drug application using the oocyte expression system, we cannot easily determine whether the movement is associated with open or desensitized states. Nevertheless, coapplication of pentobarbital and MTSEA-biotin should capture receptor states that differ from that captured by application of MTSEA-biotin alone. Concentrations of pentobarbital (500 μm) that activate the receptor slowed the rate of MTSEA-biotin modification of T160C and D163C approximately twofold but had no effect on the second-order rate constant for E155C (Table 3). Thus, T160C and D163C act as reporters of barbiturate-mediated channel gating. Concentrations of pentobarbital that do not open the channel but potentiate GABA current (e.g., 50 μm) (Table 3) slowed modification of T160C but not D163C (Fig. 7B,C; Table 3). These data suggest that movements within the binding site associated with channel gating versus allosteric modulation are distinct.

Discussion

Binding-site movements that initiate ligand-gated ion channel activation are not well established. Here, we provide evidence that movement in the β₂ Ile¹⁵⁴-Asp¹⁶³ region of the GABA_AR is involved in coupling GABA binding to channel gating, and we describe a role for β₂Glu¹⁵⁵ as an initial trigger for ion channel opening.

β₂ Ile¹⁵⁴-Asp¹⁶³ mutations affect ligand binding and channel gating

If the β₂ Ile¹⁵⁴-Asp¹⁶³ region plays a pivotal role for coupling binding to gating, one would expect that mutations within this domain would affect both processes. Seven cysteine substitutions significantly increase GABA EC₅₀ values, which reflect changes in either microscopic binding affinity and/or channel gating (Colquhoun, 1998). Three of the seven mutations that shift the GABA EC₅₀ (E155C, S156C, and G158C) also significantly reduce SR-95531 K_I, suggesting that at least one effect of these mutations is to alter ligand binding, because SR-95531 does not gate the channel (but see Ueno et al., 1997). α₁β₂(G158C) and α₁β₂(D163C) receptors have significantly reduced Hill coefficients for GABA activation, consistent with a reduction in gating efficacy (Colquhoun, 1998). β₂E155C results in spontaneously open GABA_ARs, clearly demonstrating that this mutation alters channel gating. In addition, expression of β₂E155C differentially shifts the concentration dependencies rightward for GABA, SR-95531, and P4S, indicating that the mutation also perturbs ligand binding (Zhang et al., 1994). Furthermore, tethering thiol-reactive groups onto β₂E155C closes the spontaneously open channels. Together, the data suggest that β₂Glu¹⁵⁵ occupies a key position in the activation pathway involved in coupling ligand binding site to channel gating. Although detailed kinetic analyses of these mutations are required to quantitatively tease apart the effects of each of these mutations on microscopic binding affinity and channel gating properties, the above results indicate that mutations in β₂ Ile¹⁵⁴-Asp¹⁶³ region of the GABA-binding site disrupt both affinity and efficacy.

Structural rearrangements during gating transitions

The β₂ subunit forms the principal side of the GABA-binding site. We conclude that β₂Asp¹⁶³ and β₂Thr¹⁶⁰ are found within or near the GABA-binding site, based on a slowing of the rate of MTSEA-biotin modification of T160C and D163C by both GABA and SR-95531. Protection by both ligands suggests steric hindrance of MTSEA-biotin modification, because agonists and antagonists promote different conformational changes within the binding site (Armstrong and Gouaux, 2000). In addition, mutagenesis and homology modeling studies suggest that the β₂ Ile¹⁵⁴- Asp¹⁶³ region lines part of the GABA-binding site (Amin and Weiss, 1993).

Tierney et al. (1996) predict that movements within the β₂ subunit are critical for channel gating. To test the hypothesis that the β₂ Ile¹⁵⁴-Asp¹⁶³ region of the binding site undergoes structural rearrangements during channel activation, we measured the rate of MTSEA-biotin modification of introduced cysteines in the presence of pentobarbital (500 μm). Although the binding sites for GABA and pentobarbital differ, the final structure of the activated GABA_A receptor channel is likely similar because both drugs produce similar single channel conductances (Jackson et al., 1982; Akk and Steinbach, 2000). We can therefore monitor pentobarbital-induced movements in the GABA-binding site during state transitions from resting to open-desensitized states. Coapplication of pentobarbital and MTSEA-biotin should capture this region of the receptor in a conformation that differs from that captured by application of MTSEA-biotin alone. The observation that pentobarbital significantly slows modification of T160C and D163C (approximately twofold) indicates that the environment surrounding these residues changes and that they are “conformationally sensitive” to channel activation, supporting our hypothesis that movements within this region are critical for channel gating. T160C also appears to act as a reporter for movements that occur during allosteric modulation, because the rate of MTSEA-biotin modification of T160C was slowed almost twofold in the presence of a low concentration of pentobarbital (50 μm) that potentiates but does not gate the channel.

β₂ Ile¹⁵⁴-Asp¹⁶³ is a protein hinge

Local agonist-induced movements within LGIC binding sites precede a conformational wave that leads to channel gating (Chakrapani et al., 2004). Structural studies suggest that the binding of ACh induces a 15° clockwise rotation of the inner β-sheets of the N-terminal ligand binding domain of the nAChR α₁ subunits. This, in turn, brings the β1-β2 loop (loop 2) into contact with the extracellular M2-M3 loop, and movement of the M2-M3 loop then causes the M2 region to rotate, which leads to opening of the channel gate (Miyazawa et al., 2003). Linear free energy analysis of the nAChR suggest that a conformational wave begins at the binding site and region A of the ACh-binding site (β-strand 4 and loop 5), followed by movements of loops 2, 7 (Cys-Cys loop) and the M2-M3 linker at the extracellular juxtapore region, and finally movement of the transmembrane domains (Grosman et al., 2000; Chakrapani et al., 2004). Studies examining the structural mechanisms of GABA_AR activation have identified pairs of interacting residues within these regions that are necessary for coupling GABA binding to channel gating-desensitization. These include electrostatic interactions between negatively charged residues in loops 2 (α₁Asp⁵⁷) and 7 (α₁Asp¹⁴⁹) of the GABA_AR and a positively charged lysine (α₁Lys²⁷⁹) in the M2-M3 loop (Kash et al., 2003). Recently, a study using a chimeric receptor comprised of AChBP fused to the transmembrane pore domain of the 5-HT_3A receptor demonstrated that only when loops 2, 7, and 9 (region F of the binding site) from AChBP were replaced with 5-HT_3A receptor sequences did ACh binding trigger channel opening (Bouzat et al., 2004). This indicates that loops 2, 7, and 9 are critical elements involved in coupling the extracellular binding site domain to the transmembrane channel gating domain. During examination of homology models of the GABA_AR, we noticed that the β₂ Ile¹⁵⁴-Asp¹⁶³ region of the binding site (β-strand 7 and loop 8) physically links loops 2 and 9. Thus, we speculate that the β₂ Ile¹⁵⁴-Asp¹⁶³ region may act as a protein hinge and that structural rearrangements within this region of the binding site could therefore be an efficient means for simultaneously propagating movements to both loops 2 and 9.

Movements in region B are also likely to be transmitted to the region C of the GABA-binding site (i.e., end of β-strand 9, loop 10 and beginning of β-strand 10). Based on homology models, possible interactions within regions B and C include the following amino acid pairs: β₂Glu¹⁵³ and β₂Lys¹⁹⁶, β₂Glu¹⁵⁵ and β₂Arg²⁰⁷, as well as β₂Glu¹⁶⁵ and β₂Lys¹⁹⁷. Previously, we demonstrated that β₂Arg²⁰⁷ stabilizes GABA binding (Wagner and Czajkowski, 2001; Wagner et al., 2004). We predict that the carboxylate side chain of Glu¹⁵⁵ is within 2.5 Å from the guanido group of β₂Arg²⁰⁷ (Fig. 9) and may play a role in positioning the β₂Arg²⁰⁷ side chain. This may explain why mutation of β₂Glu¹⁵⁵ disrupts orthosteric ligand binding. However, mutation of β₂Glu¹⁵⁵ also produces spontaneously open channels and indicates that perturbation of this residue has additional long-range allosteric effects that are likely propagated to the channel gate by changes in the positions of regions B and C located in the binding site as well as loops 2 and 9 near the juxtapore region. Additional experiments are needed to test these hypotheses. Support for interactions between regions B and C of LGIC binding sites comes from studies of the nAChR. It has been reported that a hydrogen bond between a residue in region B (G152K) and in region C (P193I) of the nAChR α₇ subunit is important for nAChR activation (Grutter et al., 2003) and may serve to explain how the α₁G153S human polymorphism gives rise to a slow channel myasthenic syndrome (Sine et al., 1995).

Finally, the β₂ Ile¹⁵⁴-Asp¹⁶³ region may also be involved in intersubunit interactions. Models of the N-terminal domains of GABA_AR predict that β₂Asp¹⁶³ forms a salt bridge with α₁Arg¹¹⁹ and β₂Arg²⁸. The roles of salt bridges in GABA_AR function are not presently known, but it is believed that salt bridges may limit the number of conformations of a protein complex, be key participants in determining ligand binding geometry, or be important for the association of subunits in multiprotein complexes (Hendsch and Tidor, 1994). The disruption or formation of bonds among these charged residues at subunit interfaces may be important for conformational changes that occur during activation and/or desensitization. Mutation of β₂Asp¹⁶³ significantly decreased the Hill coefficient for GABA activation of the receptor and is consistent with this hypothesis.

Conclusions

The β₂ Ile¹⁵⁴-Asp¹⁶³ region of the GABA_AR-binding site appears to be a protein hinge that is uniquely positioned to transduce binding site movements to the cascade of events that lead to opening of the ion channel. We demonstrate that the region undergoes conformational rearrangements during pentobarbital-mediated gating events, and mutation of β₂Glu¹⁵⁵ gives rise to spontaneously open channels, suggesting that movements in this region of the GABA-binding site are one of the initial triggers for coupling binding to gating. Ultimately, precise mapping of inter-residue contacts will be required to test this activation mechanism and to define the pathway leading from the binding site to the channel gate.