Stoichiometry of a Recombinant GABAA Receptor

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Volume 16, Number 17, Issue of September 1, 1996 pp. 5415-5424
Copyright ©1996 Society for Neuroscience

Stoichiometry of a Recombinant GABA_A Receptor

Yongchang Chang, Ruoping Wang, Sonal Barot, and David S. Weiss
Neurobiology Research Center, Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABA_A receptor/pore complex is presumed to be a pentamer typically composed of a combination of $alpha$ , $beta$ , and $gamma$ subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABA_A receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat $alpha$ 1( $alpha$ L263S), $beta$ 2( $alpha$ L259S), and $gamma$ 2( $alpha$ L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., $alpha$ and $alpha$ L263S), along with their wild-type counterparts (e.g., $beta$ and $gamma$ ), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class ( $alpha$ , $beta$ , and $gamma$ ) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant $alpha$ 1 $beta$ 2 $gamma$ 2 GABA_A receptor is a pentamer composed of two $alpha$ subunits, two $beta$ subunits, and one $gamma$ subunit.
Key words: GABA; stoichiometry; receptor; ion channel; mutagenesis

INTRODUCTION
The release of GABA from presynaptic nerve terminals in the CNS inhibits the postsynaptic neuron by gating a chloride-selective ion pore that is an integral component of the receptor complex. Four different classes of GABA_A receptor subunits have thus far been identified in the mammalian brain: $alpha$ , $beta$ , $gamma$ , and $delta$ (Schofield et al., 1987; Khrestchatisky et al., 1989; Lolait et al., 1989; Shivers et al., 1989; Ymer et al., 1989; Harvey et al., 1993). In addition, multiple isoforms have been isolated for each of the $alpha$ , $beta$ , and $gamma$ subunit classes. Exogenous expression of various combinations of subunits suggests that GABA_A receptors must contain $alpha$ , $beta$ , and $gamma$ subunits to reconstitute the major features of native GABA_A receptors (Levitan et al., 1988; Pritchett et al., 1989; Malherbe et al., 1990; Sigel et al., 1990; Verdoorn et al., 1990). Although structure-function studies of recombinant receptors have elucidated subunit domains and residues critical for activation and modulation (Pritchett and Seeburg, 1991; Sigel et al., 1992; Wieland et al., 1992; Amin and Weiss, 1993), the stoichiometry of the GABA_A receptor has been elusive.
Mutation of a conserved leucine in the putative second membrane-spanning domain of homomeric neuronal nicotinic acetylcholine (nACh) and serotonin type 3 (5-HT₃) receptors induced an increase in agonist sensitivity (Revah et al., 1991; Yakel et al., 1993). (nACh and 5-HT₃ receptors are members of the same ligand-operated ion channel superfamily as the GABA_A receptor.) More recent studies of heteromeric nACh receptors determined that each subunit of the receptor complex carrying this leucine mutation contributed an $approx$ 10-fold increase in ACh sensitivity (Filatov and White, 1995; Labarca et al., 1995).

We have used this leucine mutation to infer the number of each subunit type that comprises recombinant $alpha$ 1 $beta$ 2 $gamma$ 2 GABA receptors expressed in Xenopus oocytes. Mutation of this leucine to serine in either the rat $alpha$ 1, $beta$ 2, or $gamma$ 2 GABA subunit isoforms increased the sensitivity to GABA. Coexpression of wild-type and mutant subunits of each class (e.g., $alpha$ and $alpha$ -mutant), along with their wild-type counterparts (e.g., $beta$ and $gamma$ ), resulted in mixed populations of receptors with distinct GABA sensitivities. We were then able to infer the number of each subunit type from the number of components comprising these compound GABA dose-response relationships. Using this approach, we conclude that the $alpha$ 1 $beta$ 2 $gamma$ 2 GABA receptor is a pentamer composed of two $alpha$ subunits, two $beta$ subunits, and one $gamma$ subunit.

MATERIALS AND METHODS
Site-directed mutagenesis and in vitro transcription. The cDNAs were isolated by the PCR (Saiki et al., 1988) as described previously (Amin et al., 1994). The cDNAs were cloned into the pALTER vector for oligonucleotide-mediated site-directed mutagenesis using Altered Sites (Promega, Madison WI). The oligonucleotides (complementary to the sense strand) used to make the leucine to serine substitutions were $alpha$ 1(L263S): 5 $'$ -CAA GGT TGT CAT GGT ACT AAC GGT CGT CAC TCC-3 $'$ ; $beta$ 2(L259S): 5 $'$ -GAT TGT GGT CAT CGT ACT GAC AGT TGT AAT TCC-3 $'$ ; and $gamma$ 2(L274S): 5 $'$ -GAG AGT GGT CAT CGT ACT GAC AGT CGT GAT TCC-3 $'$ .
The mismatched base pairs are indicated in bold. Successful mutagenesis was confirmed by sequencing (Sanger et al., 1977). cDNAs were linearized with SspI, which leaves a several hundred base pair tail that may increase cRNA stability in the oocyte. cRNA was transcribed from the linearized cDNAs by standard in vitro transcription procedures. Methods used to match the cRNA concentrations of the three subunits have been described previously (Amin and Weiss, 1996).

Oocyte injection. Xenopus laevis (Xenopus I, Ann Arbor, MI) were anesthetized by hypothermia, and ovarian lobes were surgically removed from the frog and placed in a solution that consisted of (in mM): 82.5 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl₂, 1 MgCl₂, and 10 Na₂HPO₄, 50 U/ml penicillin, and 50 µg/ml streptomycin, pH 7.5. Oocytes were dispersed in this same solution minus CaCl₂ plus 0.3% Collagenase A (Boehringer Mannheim, Indianapolis, IN). After isolation, the oocytes were rinsed thoroughly. Stage VI oocytes were separated and maintained overnight at 18°C.

Micropipettes for injecting cRNA were fabricated on a Sutter P87 horizontal puller, and the tips were cut off with microscissors. The wild-type and mutant cRNAs were then mixed at the desired ratios and diluted with diethyl pyrocarbonate-treated water. Except for the experiments presented in Figure 5, the $alpha$ : $beta$ : $gamma$ cRNA injection ratio was always 1:1:1. The cRNA mixture was then drawn up into the micropipette and injected with the Nanoject injector (Drummond Scientific, Broomall, PA).
Fig. 5. The stoichiometry does not depend on the $alpha$ $beta$ $gamma$ cRNA injection ratio. The $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ $gamma$ _m cRNA combinations were injected into oocytes at ratios of 4:1:1 and 1:1:4, respectively. The GABA dose-response relationships were then fit by the Hill equation and compared with those determined with 1:1:1 cRNA injections. The EC₅₀ values of the GABA dose-response relationships for $alpha$ _m $beta$ $gamma$ cRNA ratios of 1:1:1 and 4:1:1 were (mean ± SD) 0.30 ± 0.13 µM (n = 9) and 0.30 ± 0.04 µM (n = 5), respectively. The EC₅₀ values of the GABA dose-response relationships for $alpha$ $beta$ $gamma$ _m cRNA ratios of 1:1:1 and 1:1:4 were 0.99 ± 0.23 µM (n = 4) and 0.98 ± 0.062 µM (n = 4), respectively. These data indicate that at least at these cRNA ratios, the number of $alpha$ and $gamma$ subunits in the GABA receptor is fixed.
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Electrophysiological recording. One to three days after injection, oocytes were placed on a 300 µm nylon mesh suspended in a small volume chamber (<100 µl). The chamber and perfusion system, which allows up to 18 different solutions to be introduced to an individual oocyte, has been described previously (Amin et al., 1994). The oocyte was perfused continuously with a solution that consisted of (in mM): 92.5 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl₂, and 1 MgCl₂, pH 7.5, and briefly switched to the test solution containing GABA.

Recording microelectrodes were fabricated with a P87 Sutter horizontal puller and filled with 3 M KCl. They had resistances of 1-3 M $Omega$ . Standard two-electrode voltage-clamp techniques were used to record currents in response to the application of GABA. In all cases, the membrane potential was clamped to $-$ 70 mV. Data were played out on a chart recorder during the experiment and recorded on tape for off-line analysis.

Peak current amplitudes were measured directly from the chart record or from the computer screen. To quantify the agonist sensitivity, each dose-response relationship was fitted with the following equation using a nonlinear least-squares method:

where x is the number of fitted components and can vary from 1 to 3, I is the peak current at a given concentration of GABA (A), I_max is the maximum current, EC₅₀ is the concentration of GABA yielding a current half the maximum, and n is the Hill coefficient.

RESULTS

Mutation of the conserved leucine in $alpha$ 1, $beta$ 2, or $gamma$ 2 increases the GABA sensitivity
The isoforms $alpha$ 1, $beta$ 2, and $gamma$ 2 are widely distributed in the brain (Benke et al., 1991; Wisden et al., 1992; Ruano et al., 1994), so we selected the $alpha$ 1 $beta$ 2 $gamma$ 2 combination for this particular study. Given the remarkable sequence homology between isoforms within a class, the conclusions we reach will likely apply to recombinant GABA receptors composed of other $alpha$ , $beta$ , and $gamma$ isoforms. In this manuscript, we designate the rat $alpha$ 1, $beta$ 2, and $gamma$ 2 subunit isoforms as simply $alpha$ , $beta$ , and $gamma$ .
A conserved leucine in the putative second membrane-spanning domain (TM2) was mutated to serine in the $alpha$ ( $alpha$ L263S), $beta$ ( $beta$ L259S), and $gamma$ ( $gamma$ L274S) subunits. (Henceforth, the subunits with the leucine mutation will be designated $alpha$ _m, $beta$ _m, and $gamma$ _m). cRNA was in vitro transcribed for both mutant and wild-type $alpha$ , $beta$ , and $gamma$ subunits, mixed in the combinations $alpha$ $beta$ $gamma$ , $alpha$ _m $beta$ $gamma$ , $alpha$ $beta$ _m $gamma$ , and $alpha$ $beta$ $gamma$ _m, and injected into Xenopus laevis oocytes. Figure 1A shows examples of GABA-activated currents from each of the four combinations of subunits. Note that much lower concentrations of GABA were required to activate the GABA receptors containing either an $alpha$ _m, $beta$ _m, or $gamma$ _m subunit. Figure 1B shows average GABA dose-response relationships for all four subunit combinations and illustrates the leftward shifts in the GABA dose- response relationships induced by the leucine to serine substitutions. The continuous lines are the best fits of the Hill equation to the data. The EC₅₀ values (concentration of GABA required for half-maximal activation) determined from the fits were (mean ± SD) $alpha$ $beta$ $gamma$ , 45.8 ± 3.6 µM; $alpha$ _m $beta$ $gamma$ , 0.30 ± 0.13 µM; $alpha$ $beta$ _m $gamma$ , 0.035 ± 0.004 µM; and $alpha$ $beta$ $gamma$ _m, 0.99 ± 0.23 µM.
Fig. 1. Mutation of the conserved leucine in TM2 of the $alpha$ 1(L263S), $beta$ 2(L259S), and $gamma$ 2(L274S) subunit increases the GABA sensitivity of the GABA_A receptor. A, GABA-activated currents from oocytes expressing $alpha$ $beta$ $gamma$ , $alpha$ _m $beta$ $gamma$ , $alpha$ $beta$ _m $gamma$ , and $alpha$ $beta$ $gamma$ _m subunit combinations. The subscript ``m'' indicates that the conserved leucine in TM2 was mutated to serine. GABA was bath-applied at the indicated concentrations. Note the increase in GABA sensitivity induced by the mutation in each subunit. Calibration: 100 sec; 350, 65, 35, and 500 nA for the four rows of traces, respectively. B, Average GABA dose-response relationships for each of the four combination of subunits (mean ± SEM). The continuous lines are the best fit of the Hill equation to the data points. The EC₅₀ values and Hill coefficients (mean ± SD) for the fits are $alpha$ $beta$ $gamma$ : 45.8 ± 3.6 µM, 1.59 ± 0.09 (n = 5); $alpha$ _m $beta$ $gamma$ : 0.30 ± 0.050 µM, 0.85 ± 0.10 (n = 9); $alpha$ $beta$ _m $gamma$ : 0.035 ± 0.004 µM, 1.12 ± 0.04 (n = 3); and $alpha$ $beta$ $gamma$ _m: 0.99 ± 0.23 µM, 1.78 ± 0.36 (n = 4).
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Studies of heteromeric nACh receptors, in which the stoichiometry has been established (Reynolds and Karlin, 1978; Lindstrom et al., 1979; Raftery et al., 1980), concluded that each subunit of the receptor complex carrying this leucine mutation contributed an $approx$ 10-fold increase in ACh sensitivity (Filatov and White, 1995; Labarca et al., 1995). For example, if two subunits in the nACh receptor complex are mutated, an $approx$ 100-fold increase in ACh sensitivity was observed. In this study, we are attempting to work in the other direction; that is, infer the number of each subunit type comprising the GABA_A receptor from the shift in GABA sensitivity induced by the mutations. The data presented in Figure 1 demonstrate a 153-, 1308-, and 46-fold decrease in the EC₅₀ with $alpha$ _m, $beta$ _m, and $gamma$ _m, respectively. Because the effects of the mutations may not be equivalent for $alpha$ , $beta$ , and $gamma$ subunits, we could not ascertain directly the stoichiometry from these observed shifts in GABA sensitivity. Similarly, the shift in ACh sensitivity for the nACh receptor with one mutant subunit varied from 13-fold to 49-fold depending on which particular subunit ( $alpha$ , $beta$ , $gamma$ , or $delta$ ) carried the mutation (Labarca et al., 1995). We now describe a strategy to ascertain the stoichiometry that is not compromised by differences in the magnitude of the shifts induced by $alpha$ _m, $beta$ _m, and $gamma$ _m.

Coexpression of wild-type and mutant subunits
Predictions Assume there is only one $alpha$ subunit in the GABA_A receptor complex. Coexpression of both $alpha$ and $alpha$ _m subunits along with wild-type $beta$ and $gamma$ subunits would result in GABA dose-response relationships having two components (Fig. 2A): one component from activation of the $alpha$ _m $beta$ $gamma$ receptors and one component from activation of the $alpha$ $beta$ $gamma$ receptors. Furthermore, on the basis of the data presented in Figure 1, the GABA EC₅₀ values of these two components would be $approx$ 0.30 and $approx$ 46 µM, respectively. Alternatively, if the GABA receptor complex contained two $alpha$ subunits, the GABA dose-response relationships resulting from coexpression of $alpha$ _m, $alpha$ , $beta$ , and $gamma$ subunits would have three components (Fig. 2B): one component from receptors in which both $alpha$ subunits are wild type (EC₅₀ $approx$ 46 µM), one component from receptors in which both $alpha$ subunits are mutant (EC₅₀ $approx$ 0.30 µM), and one component with an intermediate GABA sensitivity from receptors with one wild-type and one mutant $alpha$ subunit. If mutation of the two $alpha$ subunits has an equivalent effect, the shift in sensitivity contributed by each $alpha$ _m subunit would be the square root of the shift in EC₅₀ observed when both $alpha$ subunits are mutant (153-fold; Fig. 1B). Thus, each $alpha$ _m subunit would contribute a 12.4-fold increase in GABA sensitivity predicting an intermediate component with an EC₅₀ of 3.7 µM. (Similar logic could be applied to arrive at the four components that would result from $alpha$ $alpha$ _m $beta$ $gamma$ coexpression, assuming three $alpha$ subunits in the receptor complex.)
Fig. 2. The number of components comprising the GABA dose-response relationship with $alpha$ $alpha$ _m $beta$ $gamma$ coexpression depends on the number of $alpha$ subunits in the GABA receptor complex. A, With one $alpha$ subunit in the GABA receptor complex, the GABA dose-response relationship from oocytes coexpressing $alpha$ , $alpha$ _m, $beta$ , and $gamma$ subunits would be composed of two components (continuous line): one component from activation of $alpha$ $beta$ $gamma$ receptors and one component from activation of $alpha$ _m $beta$ $gamma$ receptors (dashed lines, scaled to facilitate comparison with the continuous line). The EC₅₀ values of these two components would be $approx$ 0.30 and $approx$ 45.8 µM (indicated on the abscissa) as demonstrated by the data in Figure 1. The mutant $alpha$ subunit is shown shaded. B, With two $alpha$ subunits in the GABA receptor complex, the GABA dose-response relationship from oocytes coexpressing $alpha$ , $alpha$ _m, $beta$ , and $gamma$ subunits would be composed of three components (continuous line): one component from activation of $alpha$ $beta$ $gamma$ receptors, one component from activation of $alpha$ _m $beta$ $gamma$ receptors, and an intermediate component from activation of receptors containing both an $alpha$ and an $alpha$ _m subunit (dashed lines). The EC₅₀ values of the first and third components would be $approx$ 0.30 and $approx$ 45.8 µM, and the predicted EC₅₀ of the intermediate component, assuming the two $alpha$ _m subunits contribute equally to the shift, would be 3.7 µM. A similar logic could be applied to $alpha$ $beta$ $beta$ _m $gamma$ and $alpha$ $beta$ $gamma$ $gamma$ _m coexpression to determine the number of $beta$ and $gamma$ subunits, respectively.
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$alpha$ $alpha$ _m $beta$ $gamma$ coexpression Figure 3A shows the results from experiments in which $alpha$ _m, $alpha$ , $beta$ , and $gamma$ subunits were all coexpressed in the same oocyte. The resulting dose-response relationship was well described by the sum of three Hill equations (continuous line). The EC₅₀ of the first component, 0.26 ± 0.05 µM, corresponds to that determined with $alpha$ _m $beta$ $gamma$ coexpression (0.30 ± 0.13 µM), and the EC₅₀ of the third component, 36.3 ± 8.1 µM, corresponds to that of the $alpha$ $beta$ $gamma$ receptor (45.8 ± 3.6 µM). The presence of an intermediate component (EC₅₀ = 2.2 ± 0.1 µM) indicates that the GABA receptor must contain more than one $alpha$ subunit (Fig. 2B). The EC₅₀ of this intermediate component is in excellent agreement with the predicted intermediate EC₅₀ of 3.7 µM (Fig. 2B). Our interpretation of this intermediate component is that it represents the activation of receptors containing both an $alpha$ and an $alpha$ _m subunit. This interpretation is supported by the experiment shown in the inset of Figure 3A. Oocytes were injected with $alpha$ $beta$ $gamma$ cRNA; 5 d later, the same oocytes were injected with $alpha$ _m $beta$ $gamma$ cRNA. Previous studies demonstrate that the cRNA for glycine subunits (Kuhse et al., 1993) or GABA subunits (Amin and Weiss, 1996) injected into Xenopus oocytes is degraded within 2-3 d. Therefore, after the second injection, the oocytes should contain only $alpha$ $beta$ $gamma$ and $alpha$ _m $beta$ $gamma$ receptors, and no intermediate component (attributable to $alpha$ $alpha$ _m $beta$ $gamma$ receptors) should be observed. This was indeed the case, because the dose-response relationship in these delayed-injection experiments was described by the sum of two Hill equations (continuous line) with EC₅₀ values of (mean ± SD) 0.42 ± 0.17 µM and 30.0 ± 7.5 µM (n = 3).
Fig. 3. GABA dose-response relationships from coexpression of $alpha$ $alpha$ _m $beta$ $gamma$ , $alpha$ $beta$ $beta$ _m $gamma$ , and $alpha$ $beta$ $gamma$ $gamma$ _m subunits. A, cRNA encoding for $alpha$ , $alpha$ _m, $beta$ , and $gamma$ subunits was coinjected into oocytes. The dose-response relationship for GABA (mean ± SEM, n = 4) was well described by the sum of three Hill equations (continuous line), suggesting, in terms of GABA sensitivity, three receptor subtypes. The EC₅₀ values of the three components were 0.26 ± 0.05, 2.2 ± 0.1, and 36.3 ± 8.1 µM ( $alpha$ -to- $alpha$ _m cRNA injection ratio = 1:3). The dashed lines representing the first component and the combination of the first and second component are shown to delineate the individual components. The three components suggest that there are two $alpha$ subunits in the GABA_A receptor complex. The inset is a GABA dose-response relationship from an oocyte expressing primarily $alpha$ $beta$ $gamma$ and $alpha$ _m $beta$ $gamma$ receptors. This was achieved by injecting $alpha$ $beta$ $gamma$ cRNA, waiting 5 d, and then injecting $alpha$ _m $beta$ $gamma$ cRNA. This ensures that $alpha$ and $alpha$ _m cRNAs do not coexist to any appreciable extent in the oocyte. In this case there was no intermediate component, and the GABA dose-response relationship was described by the sum of two Hill equations with EC₅₀ values of 0.34 and 33.4 µM (indicated by arrows). The EC₅₀ values from three such experiments (mean ± SD) were 0.42 ± 0.17 and 30.0 ± 7.5 µM for the first and second components, respectively. B, cRNA encoding for $alpha$ , $beta$ , $beta$ _m, and $gamma$ subunits was coinjected into oocytes. The dose-response relationship for GABA (mean ± SEM, n = 3) was also well described by the sum of three Hill equations (continuous line). The EC₅₀ values of the three components were 0.025 ± 0.01, 0.94 ± 0.07, and 39.2 ± 7.9 µM ( $beta$ -to- $beta$ _m cRNA injection ratio = 1:1). The three components suggest that there are two $beta$ subunits in the GABA_A receptor complex. C, cRNA encoding for $alpha$ , $beta$ , $gamma$ , and $gamma$ _m subunits was coinjected into oocytes. The dose-response relationship for GABA (mean ± SEM, n = 3) was described by the sum of two Hill equations (continuous line) with EC₅₀ values of 1.09 ± 0.12 and 40.9 ± 4.8 µM ( $gamma$ -to- $gamma$ _m cRNA injection ratio = 1:1). The dashed line represents the first component. Two components suggest that there is one $gamma$ subunit in the GABA_A receptor complex.
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$alpha$ $beta$ $beta$ _m $gamma$ coexpression Figure 3B presents the average GABA dose-response relationship from oocytes coexpressing $beta$ and $beta$ _m subunits along with wild-type $alpha$ and $gamma$ subunits. The continuous line in Figure 3B is the best fit of the sum of three Hill equations. The EC₅₀ of the first component, 0.025 ± 0.01 µM, corresponds to that determined with $alpha$ $beta$ _m $gamma$ coexpression (0.035 ± 0.004 µM), and the EC₅₀ of the third component, 39.2 ± 7.9 µM, corresponds to that of the $alpha$ $beta$ $gamma$ receptor (45.8 ± 3.6 µM). Again, the presence of an intermediate component (EC₅₀ = 0.94 ± 0.07 µM) indicates that the GABA receptor contains more than one $beta$ subunit (Fig. 2B). Assuming two $beta$ subunits and the shift observed in Figure 1B for $alpha$ $beta$ _m $gamma$ coexpression (1308-fold), each $beta$ _m subunit would contribute a 36.2-fold increase in GABA sensitivity, predicting an intermediate EC₅₀ of 1.27 µM. This is in excellent agreement with the observed intermediate EC₅₀ of 0.94 ± 0.07 µM. $alpha$ $beta$ $gamma$ $gamma$ _m coexpression The data presented thus far (Fig. 3A,B) suggest the presence of two $alpha$ subunits and two $beta$ subunits. If, by analogy with other members of this receptor-operated super-family, we assume that the GABA_A receptor is a pentameric complex (Langosch et al., 1988; Anand et al., 1991; Cooper et al., 1991; Unwin, 1993; Nayeem et al., 1994; Macdonald and Olsen, 1994), we can assume there is only one $gamma$ subunit. This inferred presence of one $gamma$ subunit is confirmed by the $alpha$ $beta$ $gamma$ $gamma$ _m coexpression studies presented in Figure 3C. In contrast to that of $alpha$ $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ $beta$ _m $gamma$ coexpression, the $alpha$ $beta$ $gamma$ $gamma$ _m dose-response relationships were described by the sum of only two Hill equations (continuous line). Furthermore, the EC₅₀ of the first component, 1.09 ± 0.12 µM, corresponds to that determined with $alpha$ $beta$ $gamma$ _m coexpression (0.99 ± 0.23), and the EC₅₀ of the second component, 40.9 ± 4.8 µM, corresponds to that determined for $alpha$ $beta$ $gamma$ receptors (45.8 ± 3.6 µM). Therefore, the two components in Figure 3C represent the activation of receptors containing either a $gamma$ or $gamma$ _m subunit, and the GABA receptor complex contains only one $gamma$ subunit.
Varying the ratio of wild-type and mutant subunits
We next considered the possibility that there are multiple indistinguishable intermediate components in the dose-response relationships of Figure 3. To address this issue, the ratio of wild-type and mutant cRNA (e.g., $alpha$ and $alpha$ _m) coinjected with their wild-type counterparts (e.g., $beta$ and $gamma$ ) was varied. For example, if the intermediate component of the GABA dose-response relationship in Figure 3A consists of more than one component, then varying the $alpha$ -to- $alpha$ _m cRNA ratio should vary the relative fractions of the different receptor combinations underlying the intermediate components and shift the apparent EC₅₀ of the intermediate component. A similar argument would apply for changes in the $beta$ -to- $beta$ _m and $gamma$ -to- $gamma$ _m cRNA ratio.
At all cRNA injection ratios tested, the GABA dose-response relationships consisted of three components for $alpha$ $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ $beta$ _m $gamma$ coexpression and two components for $alpha$ $beta$ $gamma$ $gamma$ _m coexpression. Figure 4 presents plots of the EC₅₀ values versus the fraction of the wild-type component for each of the three coexpression experiments. The fraction of the wild-type component is the amplitude of the wild-type component divided by the total amplitude and is determined from the fits to the individual compound dose-response relationships. The EC₅₀ values and component fractions are presented in Table 1. The relative fraction of the components varied in a manner that would be expected for the different cRNA injection ratios; that is, the amplitude of the wild-type component increased with an increase in the ratio of $alpha$ -to- $alpha$ _m cRNA. Figure 4A is a plot of the EC₅₀ values of the three components versus the fraction of the wild-type component for $alpha$ $alpha$ _m $beta$ $gamma$ coexpression. The dashed lines represent the mean EC₅₀ determined from expression of $alpha$ $beta$ $gamma$ and $alpha$ _m $beta$ $gamma$ (Fig. 1B). The EC₅₀ values of the first and third components do not depend on the relative proportions of the different receptor combinations consistent with the interpretation that they represent the activation of receptors containing all mutant and all wild-type $alpha$ subunits, respectively. The continuous line is from a linear regression to the EC₅₀ of the intermediate component and demonstrates a very slight, but significant (p < 0.05), negative dependence on the fraction of the wild-type component. That is, the EC₅₀ decreased slightly with increasing availability of the wild-type $alpha$ subunit. If the intermediate component was the combination of more than one subcomponent, the expected relationship should be in the opposite direction than that observed; that is, the EC₅₀ should increase with an increase in the fraction of the wild-type component. The slight decrease in the EC₅₀ with increasing fraction of the wild-type component may reflect differences in the ability to extract the EC₅₀ values from these compound dose-response relationships as the amplitudes of the components vary. Figure 4B,C are similar plots of the EC₅₀ values as a function of the fraction of the wild-type component with $alpha$ $beta$ $beta$ _m $gamma$ and $alpha$ $beta$ $gamma$ $gamma$ _m coexpression. In both cases, the EC₅₀ values of all components appeared independent of the component amplitudes (Table 1). The amplitudes of the components in these wild-type and mutant coexpression experiments did not correspond in all cases to that predicted by the binomial hypothesis. Possible explanations for this discrepancy will be considered in the Discussion.
Fig. 4. The EC₅₀ values of the components with $alpha$ $alpha$ _m $beta$ $gamma$ , $alpha$ $beta$ $beta$ _m $gamma$ , and $alpha$ $beta$ $gamma$ $gamma$ _m coexpression do not depend on the relative amplitudes of the components. A, Plot of the EC₅₀ values of the three components as a function of the fraction of the wild-type component for $alpha$ $alpha$ _m $beta$ $gamma$ coexpression. The fraction of the wild-type component is the amplitude of the wild-type component divided by the total amplitude and is determined from the fits to the individual compound dose-response relationships. In these experiments, GABA dose-response relationships were constructed from oocytes in which the $alpha$ -to- $alpha$ _m cRNA injection ratio was varied (1:3, 1:1, and 3:1) to shift the relative amplitudes of the different components. The total amount of $alpha$ and $alpha$ _m cRNA remained fixed with respect to $beta$ and $gamma$ . For all $alpha$ -to- $alpha$ _m ratios tested, the GABA dose-response relationships were described by the sum of three Hill equations. The dashed lines represent the mean EC₅₀ values determined from $alpha$ $beta$ $gamma$ and $alpha$ _m $beta$ $gamma$ coexpression (Fig. 1). The continuous line is a linear regression to the EC₅₀ of the intermediate component and demonstrates a slight but significant (p < 0.05) negative dependence that is in the opposite direction from that predicted if there were multiple, indistinguishable, intermediate components (see text). These data indicate three components and hence two $alpha$ subunits in the GABA receptor complex. B, Similar plot as in A, but for $alpha$ $beta$ $beta$ _m $gamma$ coexpression at different ratios of $beta$ to $beta$ _m (1:1 and 3:1). The dashed lines represent the mean EC₅₀ values determined from $alpha$ $beta$ $gamma$ and $alpha$ $beta$ _m $gamma$ coexpression (Fig. 1). These data indicate three components and hence two $beta$ subunits in the GABA receptor complex. C, Similar plot as in A, but for $alpha$ $beta$ $gamma$ $gamma$ _m coexpression at different ratios of $gamma$ to $gamma$ _m (1:3 and 1:1). These data indicate two components and hence one $gamma$ subunit in the GABA receptor complex.
[View Larger Version of this Image (18K GIF file)]

Table 1. EC₅₀ values and fractions of the components for the different wild-type/mutant cRNA injection ratios

cRNA injection ratio First component
Second component
Third component
n

EC₅₀(µM) Fraction EC₅₀ (µM) Fraction EC₅₀ (µM) Fraction

$alpha$ $alpha$ _m(1:3) $beta$ $gamma$ 0.26 ± 0.05 0.43 ± 0.08 2.2 ± 0.1 0.24 ± 0.13 36.3 ± 8.1 0.33 ± 0.15 4

$alpha$ $alpha$ _m(1:1) $beta$ $gamma$ 0.41 ± 0.08 0.17 ± 0.05 2.0 ± 0.2 0.10 ± 0.02 42.4 ± 10.4 0.72 ± 0.07 3

$alpha$ $alpha$ _m(3:1) $beta$ $gamma$ 0.35 ± 0.17 0.05 ± 0.04 2.0 ± 0.1 0.09 ± 0.02 40.1 ± 5.7 0.86 ± 0.01 2

$alpha$ $beta$ $beta$ _m(1:1) $gamma$ 0.025 ± 0.01 0.17 ± 0.03 0.94 ± 0.07 0.6 ± 0.04 39.2 ± 7.9 0.27 ± 0.07 4

$alpha$ $beta$ $beta$ _m(3:1) $gamma$ 0.038 ± 0.01 0.03 ± 0.01 0.92 ± 0.05 0.3 ± 0.02 43.4 ± 0.9 0.63 ± 0.02 3

$alpha$ $beta$ $gamma$ $gamma$ _m(1:3) 1.16 ± 0.14 0.58 ± 0.12 47.9 ± 19.8 0.42 ± 0.12 3

$alpha$ $beta$ $gamma$ $gamma$ _m(1:1) 1.09 ± 0.12 0.28 ± 0.12 40.9 ± 4.8 0.72 ± 0.12 3

Values are mean ± SD. In all cases, total $alpha$ : $beta$ : $gamma$ cRNA ratios were 1:1:1.

Nevertheless, the data presented in Figure 4 suggest that in terms of GABA sensitivity, there are three receptor types with $alpha$ $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ $beta$ _m $gamma$ coexpression and two receptor types with $alpha$ $beta$ $gamma$ $gamma$ _m coexpression, further supporting a pentameric GABA_A receptor composed of two $alpha$ subunits, two $beta$ subunits, and one $gamma$ subunit.

The stoichiometry does not depend on the relative availability of $alpha$ , $beta$ , and $gamma$ subunits
Finally, we considered whether the stoichiometry of the GABA receptor is fixed or can vary, depending on the relative abundance of the different subunit types. To address this issue, we injected $alpha$ _m, $beta$ , and $gamma$ cRNA at a ratio of 4:1:1 or $alpha$ , $beta$ , and $gamma$ _m cRNA at a ratio of 1:1:4. If the increased availability of $alpha$ or $gamma$ subunits can increase the number of $alpha$ or $gamma$ subunits incorporated into the GABA receptor, the EC₅₀ values should shift further to the left than that observed with equivalent cRNA injection ratios (Fig. 1B). The results from these experiments are presented in Figure 5. For both cases, the EC₅₀ values of the GABA dose-response relationships with nonequivalent injection ratios were indistinguishable from those determined with equivalent cRNA injection ratios. These data indicate that at least at these cRNA ratios, the number of $alpha$ and $gamma$ subunits in the GABA receptor is fixed. By default, the number of $beta$ subunits should be fixed as well.

DISCUSSION

Summary, assumptions, and limitations
By taking advantage of a mutation in the putative second membrane-spanning domain of the GABA_A receptor subunits that increased the GABA sensitivity in relative proportion to the number of subunits carrying the mutation, we were able to infer that recombinant $alpha$ 1 $beta$ 2 $gamma$ 2 receptors are pentamers composed of two $alpha$ subunits, two $beta$ subunits, and one $gamma$ subunit. We have also demonstrated that at least over a fourfold ratio of injected cRNA concentrations, the stoichiometry appears to be fixed. Behe et al. (1995) used a similar strategy of wild-type and mutant subunit coexpression to determine the number of incorporated NR1 subunits in recombinant N-methyl-D-aspartate (NMDA) receptors, although their approach differs from the present study in that they relied on mutation-induced alterations in the single-channel conductance.
Our conclusions are tied strongly to the assumption that the mutations do not alter the normal stoichiometry of the $alpha$ 1 $beta$ 2 $gamma$ 2 GABA_A receptor. Although we have no direct evidence, it seems unlikely that a point mutation in a residue that is presumed to face the interior of the pore (Revah et al., 1991; Unwin, 1995; Xu and Akabas, 1996) would so drastically affect subunit-subunit interactions as to induce an atypical stoichiometry. We have also assumed that the different possible subunit arrangements of the mutant and wild-type subunits within the pentameric complex are functionally equivalent.

To uncover any possible hidden components in the compound dose-response relationships of Figure 3, we varied the wild-type-to-mutant cRNA ratio for each of the subunits (e.g., $alpha$ , $alpha$ _m) and coexpressed this mixture along with their wild-type counterparts (e.g., $beta$ , $gamma$ ). Although the relative amplitudes of the components depended on the wild-type-to-mutant cRNA ratio, the observed number of components and their EC₅₀ values were essentially independent of the cRNA ratio. This supports the conclusion that the dose-response relationships in Figure 3A-C are composed of only three, three, and two components, respectively.

We do not believe that the shifts in EC₅₀ in Figure 1, and the additional components in Figure 2, result from the expression of subpopulations of GABA receptors with different GABA sensitivities (e.g., $alpha$ $beta$ , $beta$ $gamma$ , etc.) for the following reasons. (1) Expression of $alpha$ , $beta$ , or $gamma$ subunits alone, as well as $alpha$ / $gamma$ coexpression, does not yield functional GABA receptors in oocytes. (2) $beta$ $gamma$ receptors have an EC₅₀ of 30.1 ± 8.4 µM, which is similar to that of $alpha$ $beta$ $gamma$ GABA receptors (45.8 ± 3.6 µM). (3) $alpha$ $beta$ GABA receptors have an EC₅₀ of 4.2 ± 0.9 µM (Amin et al., 1994), which is less GABA-sensitive than any of the nonwild-type components (Figs. 2 and 3; Table 1). The observation that the EC₅₀ values do not shift with changes in the wild-type-to-mutant coinjection ratios (Fig. 4; Table 1) further strengthens the conclusion that subpopulations of GABA receptors cannot account for the shifts in GABA sensitivity.

The amplitudes of the components for the different cRNA injection ratios deviated somewhat from that predicted by the binomial distribution. For example, at a wild-type-to-mutant cRNA ratio of 1:1 (Table 1), the fractional amplitudes of the wild-type components were 0.72 ± 0.07 (n = 3), 0.27 ± 0.07 (n = 4), and 0.72 ± 0.12 (n = 3) for $alpha$ $alpha$ _m $beta$ $gamma$ , $alpha$ $beta$ $beta$ _m $gamma$ , and $alpha$ $beta$ $gamma$ $gamma$ _m, respectively. Assuming that the percentages of the different receptor combinations were to follow a binomial distribution, the fraction of wild-type receptors should be 0.25 for $alpha$ $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ $beta$ _m $gamma$ coexpression and 0.50 for $alpha$ $beta$ $gamma$ $gamma$ _m coexpression. Several factors could account for this discrepancy. First, the mutations may affect subunit assembly. If the mutant subunit does not assemble as efficiently as that of the wild type, the amplitudes of the components would deviate from those predicted by the binomial distribution. Second, if the mutation altered the maximum percent time open or the single-channel conductance, the relative amplitudes of the components would be altered accordingly. In fact, the mutations do affect the Hill coefficients slightly (see Fig. 1 legend), suggesting an alteration in the gating kinetics. Third, the determination of the amplitudes of the components may be influenced by receptor desensitization. At high agonist concentrations, desensitization would diminish the relative contribution of the receptors in the more GABA-sensitive components, thus depressing the apparent amplitude of the wild-type component. Fourth, in the case of $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ _m $gamma$ coexpression, we observed a picrotoxin-sensitive current in the absence of GABA that was not observed in oocytes expressing $alpha$ $beta$ $gamma$ _m or $alpha$ $beta$ $gamma$ receptors (data not shown). This may represent the spontaneous opening of GABA receptors with two mutant subunits incorporated. If so, this would alter the relative contributions of the components in the compound dose-response relationship with $alpha$ $alpha$ _m $beta$ $gamma$ and $alpha$ $beta$ $beta$ _m $gamma$ coexpression. And finally, there may be slight errors in estimating the relative wild-type and mutant cRNA concentrations from the agarose gels. Although these factors could influence the estimation of the parameters in these compound dose-response relationships, it is not apparent how they would alter the general conclusions from this study, which rely more heavily on a detection of the number of components. Moreover, our conclusions are strengthened by the excellent agreement between the observed and predicted EC₅₀ values of the components in Figures 3 and 4.

Comparison with other studies
Our studies are consistent with evidence indicating that some native (Duggan et al., 1991; Luddens et al., 1991; Pollard et al., 1995) and recombinant (Verdoorn, 1994) GABA receptors can contain multiple subtypes of the $alpha$ subunit. In addition, Im et al. (1995), using tandem constructs of $alpha$ 6- $beta$ 2 GABA subunits, concluded that $alpha$ 6 $beta$ 2 $gamma$ 2 GABA receptors contain two $alpha$ subunits, two $beta$ subunits, and one $gamma$ subunit.
Our results are in contrast to those of Benke et al. (1994), which infer that only one $beta$ subunit is present in the native GABA receptor. This conclusion was reached from the observation that [³H]flumazenil or [³H]muscimol binding to native GABA receptors immunoprecipitated from brain by $beta$ 1, $beta$ 2, or $beta$ 3 antiserum sum to ~100% of the total binding. If neurons dominantly express one isoform of the $beta$ subunit, however, the results of Benke et al. (1994) would be obtained regardless of the number of $beta$ subunits in the GABA receptor complex. In fact, immunolocalization studies indicate some segregation of the $beta$ subunit isoforms to different brain regions (Benke et al., 1994).

Quirk et al. (1994) observed that the sum of the percentages of [³H]muscimol binding sites immunoprecipitated by $gamma$ 2 and $gamma$ 3 subunit antisera was slightly greater than the [³H]muscimol binding observed with $gamma$ 2 and $gamma$ 3 antisera in combination, although as the authors report, the difference was within the limit of error of the measurement. Khan et al. (1994) made a similar observation using antibodies raised to $gamma$ 2S and $gamma$ 2L subunits. In both cases, the data were used to conclude that a minor fraction of the GABA receptors can contain more than one type of $gamma$ subunit. Furthermore, GABA receptors obtained by $gamma$ 3 immunoprecipitation were labeled in a Western blot probed with $gamma$ 2 antisera, again suggesting that $gamma$ 2 and $gamma$ 3 subunits can coexist in the same GABA receptor complex. This conclusion seems to contradict our findings that the $alpha$ 1 $beta$ 2 $gamma$ 2 GABA receptor contains a single copy of the $gamma$ 2 subunit. It should be kept in mind, however, that immunoprecipitation studies investigate total GABA or benzodiazepine binding to isolated membranes. This approach may include (1) a subpopulation of nonfunctional GABA receptors having an aberrant stoichiometry (our approach is limited to functional GABA_A receptors) or (2) a subpopulation of $beta$ $gamma$ or $alpha$ $gamma$ GABA receptors that may contain multiple $gamma$ subunits. It is also possible that GABA receptors may have a stoichiometry that depends on the expression system (neurons, transfected mammalian cells, oocytes) or the particular $alpha$ , $beta$ , and $gamma$ subunit isoforms.

Finally, Backus et al. (1993) examined the stoichiometry of rat $alpha$ 3 $beta$ 2 $gamma$ 2 receptors in transfected human embryonic kidney cells (HEK293) and concluded that the possible subunit stoichiometries for this receptor were 2 $alpha$ 1 $beta$ 2 $gamma$ , 2 $alpha$ 2 $beta$ 1 $gamma$ , or 1 $alpha$ 2 $beta$ 2 $gamma$ , of which 2 $alpha$ 1 $beta$ 2 $gamma$ was favored. This study examined the degree of outward rectification induced by charged substitutions in homologous positions of the putative pore-forming domain of the $alpha$ 3, $beta$ 2, and $gamma$ 2 subunit isoforms. As stated by the authors, the conclusions assumed that the degree of the effect of changing the charge on the outward rectification is the same for all the subunits. Given that the amino acid at one of the two mutated positions was not conserved across the three subunits (arginine, tyrosine, or lysine), this assumption may not hold true. Perhaps both conservative and nonconservative substitutions (in terms of charge) at these positions, along with wild-type and mutant coexpression of each subunit type, could be used to test this assumption.

Arrangement of the subunits
The question of the precise manner in which the two $alpha$ subunits, two $beta$ subunits, and one $gamma$ subunit are arranged within the pentameric structure is still unanswered. Some insight may be provided by structure/function studies of the GABA_A receptor. For example, site-directed mutagenesis has previously identified a residue on the $alpha$ subunit and two domains on the $beta$ subunit that seem to form part of the GABA binding site (Sigel et al., 1992; Amin and Weiss, 1993). If, as for the nACh receptor, the agonist binding sites are at subunit interfaces (Kurosaki et al., 1987; Blount and Merlie, 1989; Pedersen and Cohen, 1990; Czajkowski and Karlin, 1991; Sine and Claudio, 1991; Sine, 1993), at least one of the two GABA binding sites may be at the $alpha$ - $beta$ subunit interface (Smith and Olsen, 1995). A detailed single-channel kinetic analysis (Sine and Steinbach, 1987; Colquhoun and Ogden, 1988; Weiss and Magleby, 1989; Twyman et al., 1990; Newland et al., 1991) may provide information on the equivalence of the two GABA binding sites and help place the second binding site at either the other $alpha$ - $beta$ interface or at a $beta$ - $gamma$ or $alpha$ - $gamma$ interface.
Although the potential subunit interactions underlying activation and modulation of GABA_A receptors are yet to be elucidated, knowledge of the stoichiometry will facilitate the incorporation of the wealth of structural and functional information into a cohesive model of the GABA_A receptor/pore complex.

FOOTNOTES

Received May 1, 1996; revised June 14, 1996; accepted June 18, 1996.

This research was supported by National Institutes of Health Grants AA09212 and NS35291 to D.S.W.

Correspondence should be addressed to David S. Weiss, Neurobiology Research Center, University of Alabama at Birmingham, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021.

REFERENCES

Amin J, Weiss DS (1993) GABA_A receptor needs two homologous domains of the $beta$ subunit for activation by GABA, but not by pentobarbital. Nature 366:565-569 . [Medline]
Amin J, Weiss DS (1996) Insight into the activation mechanism of $rho$ 1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits. Proc R Soc Lond [Biol] 263:273-282. [Medline]
Amin J, Dickerson IM, Weiss DS (1994) The agonist binding site of the GABA_A channel is not formed by the extracellular cysteine loop. Mol Pharmacol 45:317-323 . [Abstract]
Anand R, Conroy WG, Schoepfer R, Whiting P, Lindstrom J (1991) Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure. J Biol Chem 266:11192-11198 . [Abstract/Free Full Text]
Backus KH, Arigoni M, Drescher U, Scheurer L, Malherbe P, Mohler H, Benson JA (1993) Stoichiometry of a recombinant GABA_A receptor deduced from mutation-induced rectification. NeuroReport 5:285-288 . [Web of Science][Medline]
Behe P, Stern P, Wyllie DJA, Nassar M, Schoepfer R, Colquhoun D (1995) Determination of NMDA NR1 subunit copy number in recombinant NMDA receptor. Proc R Soc Lond [Biol] 262:205-213 . [Medline]
Benke D, Mertens S, Trzeciak A, Gillessen D, Mohler H (1991) GABA_A receptors display association of $gamma$ 2-subunit with $alpha$ 1- and $beta$ 2/3-subunits. J Biol Chem 266:4478-4483 . [Abstract/Free Full Text]
Benke D, Fritschy J-M, Trzeciak A, Bannwarth W, Mohler H (1994) Distribution, prevalence, and drug binding profile of $gamma$ -aminobutyric acid type A receptor subtypes differing in the $beta$ subunit variant. J Biol Chem 268:27100-27107. [Abstract/Free Full Text]
Blount P, Merlie JP (1989) Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron 3:349-357 . [Web of Science][Medline]
Colquhoun D, Ogden DC (1988) Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J Physiol (Lond) 395:131-159 . [Abstract/Free Full Text]
Cooper E, Couturier S, Ballivet M (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature 350:235-238 . [Medline]
Czajkowski C, Karlin A (1991) Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor: a negatively charged region of the $delta$ subunit within 0.9 nm of the alpha subunit binding site disulfide. J Biol Chem 266:22603-22612 . [Abstract/Free Full Text]
Duggan MJ, Pollard S, Stephenson FA (1991) Immunoaffinity purification of GABA_A receptor alpha-subunit iso-oligomers: demonstration of receptor populations containing $alpha$ 1, $alpha$ 2: $alpha$ 1 $alpha$ 3, and $alpha$ 2 $alpha$ 3 subunit pairs. J Biol Chem 266:24778-24784 . [Abstract/Free Full Text]
Filatov GN, White MM (1995) The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating. Mol Pharmacol 48:379-384 . [Abstract]
Harvey RJ, Kim H-C, Darlison MG (1993) Molecular cloning reveals the existence of a fourth gamma subunit of the vertebrate brain GABA_A receptor. FEBS Lett 331:211-216 . [Web of Science][Medline]
Im WB, Pregenzer JF, Binder JA, Dillon GH, Alberts GL (1995) Chloride channel expression with the tandem construct of $alpha$ 6- $beta$ 2 GABAA receptor subunit requires a monomeric subunit of $alpha$ 6 or $gamma$ 2. J Biol Chem 270:26063-26066 . [Abstract/Free Full Text]
Khan ZU, Gutierrez A, De Blas AL (1994) Short and long form $gamma$ 2 subunits of the GABA_A/benzodiazepine receptors. J Neurochem 63:1466-1476 . [Web of Science][Medline]
Khrestchatisky M, MacLennan AJ, Chiang MY, Xu WT, Jackson MB, Brecha N, Sternini C, Olsen RW, Tobin AJ (1989) A novel $alpha$ subunit in rat brain GABA receptors. Neuron 3:745-753 . [Web of Science][Medline]
Kuhse J, Laube B, Magalei D, Betz H (1993) Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry. Neuron 11:1049-1056 . [Web of Science][Medline]
Kurosaki T, Fukuda K, Konno T, Mori Y, Tanaka K, Mishina M, Numa S (1987) Functional properties of nicotinic acetylcholine receptor subunits expressed in various combinations. FEBS Lett 214:253-258 . [Web of Science][Medline]
Labarca C, Nowak MW, Zhang H, Tang L, Deshpande P, Lester HA (1995) Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature 376:514-516 . [Medline]
Langosch D, Thomas L, Betz H (1988) Conserved Quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. Proc Natl Acad Sci USA 85:7394-7398 . [Abstract/Free Full Text]
Levitan ES, Blair LAC, Dionne VE, Barnard EA (1988) Biophysical and pharmacological properties of cloned GABA_A receptor subunits expressed in Xenopus oocytes. Neuron 1:773-781 . [Web of Science][Medline]
Lindstrom J, Merlie J, Yogeeswaran G (1979) Biochemical properties of acetylcholine receptor subunits from Torpedo californica. Biochemistry 18:4465-4470 . [Medline]
Lolait SJ, O'Carroll AM, Kusano K, Muller JM, Brownstein MJ, Mahan LC (1989) Cloning and expression of a novel rat GABA receptor. FEBS Lett 246:145-148 . [Web of Science][Medline]
Luddens H, Killisch I, Seeburg PH (1991) More than one $alpha$ -variant may exist in a GABA/benzodiazepine receptor complex. J Recept Res 11:535-551 . [Web of Science][Medline]
Macdonald RL, Olsen RW (1994) GABA receptor channels. Annu Rev Neurosci 17:569-602 . [Web of Science][Medline]
Malherbe P, Sigel E, Baur R, Persohn E, Richards J, Mohler H (1990) Functional characteristics and sites of gene expression of the $alpha$ 1, $beta$ 1, $gamma$ 2-isoform of the rat GABA_A receptor. J Neurosci 10:2330-2337 . [Abstract]
Nayeem N, Green TP, Martin IL, Barnard EA (1994) Quaternary structure of the native GABA_A receptor determined by electron microscopic image analysis. J Neurochem 62:815-818 . [Web of Science][Medline]
Newland CF, Colquhoun D, Cull-Candy SG (1991) Single channels activated by high concentrations of GABA in superior cervical ganglion neurones of the rat. J Physiol (Lond) 432:203-233 . [Abstract/Free Full Text]
Pedersen SE, Cohen JB (1990) D-Tubocurarine binding sites are located at $alpha$ - $gamma$ and $alpha$ - $delta$ subunit interfaces of the nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 87:2785-2789 . [Abstract/Free Full Text]
Pollard S, Thompson CL, Stephenson FA (1995) Quantitative characterization of $alpha$ 6 and $alpha$ 1 $alpha$ 6 subunit-containing native $gamma$ -aminobutyric acid A receptors of adult rat cerebellum demonstrates two alpha subunits per receptor oligomer. J Biol Chem 270:21285-21290 . [Abstract/Free Full Text]
Pritchett DB, Seeburg PH (1991) $gamma$ -Aminobutyric acid type A receptor point mutation increases the affinity of compounds for the benzodiazepine site. Proc Natl Acad Sci USA 88:1421-1425 . [Abstract/Free Full Text]
Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H, Schofield PR, Seeburg PH (1989) Importance of a novel GABA_A receptor subunit for benzodiazepine pharmacology. Nature 338:582-585 . [Medline]
Quirk K, Gillard NP, Ragan CI, Whiting PJ, McKernan RM (1994) $gamma$ -aminobutyric acid type A receptors in the rat brain can contain both $gamma$ 2 and $gamma$ 3 subunits, but $gamma$ 1 does not exist in combination with another $gamma$ subunit. Mol Pharmacol 45:1061-1070 . [Abstract]
Raftery MA, Hunkapiller MW, Strader CD, Hood LE (1980) Acetylcholine receptor: complex of homologous subunits. Science 208:1454-1457 . [Abstract/Free Full Text]
Revah F, Bertrand D, Galzi J-L, Devillers-Thiery A, Mulle C, Hussy N, Bertrand S, Ballivet M, Changeux J-P (1991) Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353:846-849 . [Medline]
Reynolds JA, Karlin A (1978) Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Biochemistry 17:2035-2038 . [Medline]
Ruano D, Araujo F, Machado A, De Blas AL, Vitorica J (1994) Molecular characterization of type I GABA_A receptor complex from rat cerebral cortex and hippocampus. Mol Brain Res 25:225-233 . [Medline]
Saiki RK, Gelfand DH, Stoffel DH, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer directed enzyme amplification with a thermostable DNA polymerase. Science 239:487-491 . [Abstract/Free Full Text]
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 . [Abstract/Free Full Text]
Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, Seeburg PH, Barnard EA (1987) Sequence and functional expression of the GABA_A receptor shows a ligand-gated receptor super-family. Nature 328:221-227 . [Medline]
Shivers BD, Killisch I, Sprengel R, Sontheimer H, Kohler M, Schofield PR, Seeburg PH (1989) Two novel GABA_A receptor subunits exist in distinct neuronal subpopulations. Neuron 3:327-337 . [Web of Science][Medline]
Sigel E, Baur R, Trube G, Mohler H, Malherbe P (1990) The effect of subunit composition of rat brain GABA_A receptor on channel function. Neuron 5:703-711 . [Web of Science][Medline]
Sigel E, Baur R, Kellenberger S, Malherbe P (1992) Point mutations affecting antagonist affinity and agonist dependent gating of GABA_A receptor channels. EMBO J 11:2017-2023 . [Web of Science][Medline]
Sine SM (1993) Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity. Proc Natl Acad Sci USA 90:9436-9440 . [Abstract/Free Full Text]
Sine SM, Claudio T (1991) $gamma$ - and $delta$ -subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor. J Biol Chem 266:19369-19377 . [Abstract/Free Full Text]
Sine SM, Steinbach JH (1987) Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist. J Physiol (Lond) 385:325-359 . [Abstract/Free Full Text]
Smith G, B, Olsen RW (1995) Functional domains of GABA_A receptors. Trends Pharmacol Sci 16:162-168 . [Medline]
Twyman RE, Rogers CJ, Macdonald RL (1990) Intraburst kinetic properties of the GABA_A receptor main conductance state of mouse spinal cord neurones in culture. J Physiol (Lond) 423:193-220 . [Abstract/Free Full Text]
Unwin N (1993) Nicotinic acetylcholine receptors at 9 A resolution. J Mol Biol 229:1101-1124 . [Web of Science][Medline]
Unwin N (1995) Acetylcholine receptor channel imaged in the open state. Nature 373:37-43 . [Medline]
Verdoorn TA (1994) Formation of heteromeric $gamma$ -aminobutyric acid type A receptors containing two different $alpha$ subunits. Mol Pharmacol 45:475-480 . [Abstract]
Verdoorn TA, Draguhn A, Ymer S, Seeburg PH, Sakmann (1990) Functional properties of recombinant rat GABA_A receptors depend upon subunit composition. Neuron 4:919-928 . [Web of Science][Medline]
Weiss DS, Magleby KL (1989) Gating scheme for single GABA-activated chloride channels determined from stability plots, dwell-time distributions, and adjacent interval durations. J Neurosci 9:1314-1324 . [Abstract]
Wieland HA, Luddens H, Seeburg PH (1992) A single histidine in GABA_A receptors is essential for benzodiazepine agonist binding. J Biol Chem 267:1426-1429 . [Abstract/Free Full Text]
Wisden W, Laurie DJ, Monyer H, Seeburg PH (1992) The distribution of 13 GABA_A receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 12:1040-1062 . [Abstract]
Xu M, Akabas MH (1996) Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA_A receptor $alpha$ 1 subunit. J Gen Physiol 107:195-205. [Abstract/Free Full Text]
Yakel J, L, Lagrutta A, Adelman JP, North RA (1993) Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes. Proc Natl Acad Sci USA 90:5030-5033 . [Abstract/Free Full Text]
Ymer S, Schofield PR, Draguhn A, Werner P, Kohler M, Seeburg PH (1989) GABA_A receptor $beta$ subunit heterogeneity: functional expression of cloned cDNAs. EMBO J 8:1665-1670 . [Web of Science][Medline]

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A. Zeller, M. Arras, R. Jurd, and U. Rudolph
Identification of a Molecular Target Mediating the General Anesthetic Actions of Pentobarbital
Mol. Pharmacol., March 1, 2007; 71(3): 852 - 859.
[Abstract] [Full Text] [PDF]

I. Sarto-Jackson, R. Furtmueller, M. Ernst, S. Huck, and W. Sieghart
Spontaneous Cross-link of Mutated {alpha}1 Subunits during GABAA Receptor Assembly
J. Biol. Chem., February 16, 2007; 282(7): 4354 - 4363.
[Abstract] [Full Text] [PDF]

S. J. Bailey, M. A. Ravier, and G. A. Rutter
Glucose-Dependent Regulation of {gamma}-Aminobutyric Acid (GABAA) Receptor Expression in Mouse Pancreatic Islet {alpha}-Cells
Diabetes, February 1, 2007; 56(2): 320 - 327.
[Abstract] [Full Text] [PDF]

M. R. McCartney, T. Z. Deeb, T. N. Henderson, and T. G. Hales
Tonically Active GABAA Receptors in Hippocampal Pyramidal Neurons Exhibit Constitutive GABA-Independent Gating
Mol. Pharmacol., February 1, 2007; 71(2): 539 - 548.
[Abstract] [Full Text] [PDF]

A. H. Lagrange, E. J. Botzolakis, and R. L. Macdonald
Enhanced macroscopic desensitization shapes the response of {alpha}4 subtype-containing GABAA receptors to synaptic and extrasynaptic GABA
J. Physiol., February 1, 2007; 578(3): 655 - 676.
[Abstract] [Full Text] [PDF]

A. Sedelnikova, B. E. Erkkila, H. Harris, S. O. Zakharkin, and D. S. Weiss
Stoichiometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor
J. Physiol., December 1, 2006; 577(2): 569 - 577.
[Abstract] [Full Text] [PDF]

Y. Chen, K. Reilly, and Y. Chang
Evolutionarily Conserved Allosteric Network in the Cys Loop Family of Ligand-gated Ion Channels Revealed by Statistical Covariance Analyses
J. Biol. Chem., June 30, 2006; 281(26): 18184 - 18192.
[Abstract] [Full Text] [PDF]

R. Serantes, F. Arnalich, M. Figueroa, M. Salinas, E. Andres-Mateos, R. Codoceo, J. Renart, C. Matute, C. Cavada, A. Cuadrado, et al.
Interleukin-1beta Enhances GABAA Receptor Cell-surface Expression by a Phosphatidylinositol 3-Kinase/Akt Pathway: RELEVANCE TO SEPSIS-ASSOCIATED ENCEPHALOPATHY
J. Biol. Chem., May 26, 2006; 281(21): 14632 - 14643.
[Abstract] [Full Text] [PDF]

J B Vincent, S. Horike, S Choufani, A D Paterson, W Roberts, P Szatmari, R Weksberg, B Fernandez, and S W Scherer
An inversion inv(4)(p12-p15.3) in autistic siblings implicates the 4p GABA receptor gene cluster
J. Med. Genet., May 1, 2006; 43(5): 429 - 434.
[Abstract] [Full Text] [PDF]

R. E. Petroski, J. E. Pomeroy, R. Das, H. Bowman, W. Yang, A. P. Chen, and A. C. Foster
Indiplon Is a High-Affinity Positive Allosteric Modulator with Selectivity for {alpha}1 Subunit-Containing GABAA Receptors
J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 369 - 377.
[Abstract] [Full Text] [PDF]

C. M. McCann, J. Bracamontes, J. H. Steinbach, and J. R. Sanes
The cholinergic antagonist {alpha}-bungarotoxin also binds and blocks a subset of GABA receptors
PNAS, March 28, 2006; 103(13): 5149 - 5154.
[Abstract] [Full Text] [PDF]

M. M. White
Pretty Subunits All in a Row: Using Concatenated Subunit Constructs to Force the Expression of Receptors with Defined Subunit Stoichiometry and Spatial Arrangement
Mol. Pharmacol., February 1, 2006; 69(2): 407 - 410.
[Abstract] [Full Text] [PDF]

A. J. Boileau, R. A. Pearce, and C. Czajkowski
Tandem Subunits Effectively Constrain GABAA Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAA Receptor-Associated Protein
J. Neurosci., December 7, 2005; 25(49): 11219 - 11230.
[Abstract] [Full Text] [PDF]

P. V. Plazas, E. Katz, M. E. Gomez-Casati, C. Bouzat, and A. B. Elgoyhen
Stoichiometry of the {alpha}9{alpha}10 Nicotinic Cholinergic Receptor
J. Neurosci., November 23, 2005; 25(47): 10905 - 10912.
[Abstract] [Full Text] [PDF]

A. K. Bera and M. H. Akabas
Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments
J. Biol. Chem., October 21, 2005; 280(42): 35506 - 35512.
[Abstract] [Full Text] [PDF]

R. Baur, U. Simmen, M. Senn, U. Sequin, and E. Sigel
Novel Plant Substances Acting as {beta} Subunit Isoform-Selective Positive Allosteric Modulators of GABAA Receptors
Mol. Pharmacol., September 1, 2005; 68(3): 787 - 792.
[Abstract] [Full Text] [PDF]

D. M. Jones-Davis, L. Song, M. J. Gallagher, and R. L. Macdonald
Structural Determinants of Benzodiazepine Allosteric Regulation of GABAA Receptor Currents
J. Neurosci., August 31, 2005; 25(35): 8056 - 8065.
[Abstract] [Full Text] [PDF]

R. Baur and E. Sigel
Benzodiazepines Affect Channel Opening of GABAA Receptors Induced by Either Agonist Binding Site
Mol. Pharmacol., April 1, 2005; 67(4): 1005 - 1008.
[Abstract] [Full Text] [PDF]

A. Sedelnikova, C. D. Smith, S. O. Zakharkin, D. Davis, D. S. Weiss, and Y. Chang
Mapping the {rho}1 GABAC Receptor Agonist Binding Pocket: CONSTRUCTING A COMPLETE MODEL
J. Biol. Chem., January 14, 2005; 280(2): 1535 - 1542.
[Abstract] [Full Text] [PDF]

S. Jung, M. H. Akabas, and R. A. Harris
Functional and Structural Analysis of the GABAA Receptor {alpha}1 Subunit during Channel Gating and Alcohol Modulation
J. Biol. Chem., January 7, 2005; 280(1): 308 - 316.
[Abstract] [Full Text] [PDF]

J. G. Newell, R. A. McDevitt, and C. Czajkowski
Mutation of Glutamate 155 of the GABAA Receptor {beta}2 Subunit Produces a Spontaneously Open Channel: A Trigger for Channel Activation
J. Neurosci., December 15, 2004; 24(50): 11226 - 11235.
[Abstract] [Full Text] [PDF]

F. Sancar and C. Czajkowski
A GABAA Receptor Mutation Linked to Human Epilepsy ({gamma}2R43Q) Impairs Cell Surface Expression of {alpha}{beta}{gamma} Receptors
J. Biol. Chem., November 5, 2004; 279(45): 47034 - 47039.
[Abstract] [Full Text] [PDF]

J. Kang and R. L. Macdonald
The GABAA Receptor {gamma}2 Subunit R43Q Mutation Linked to Childhood Absence Epilepsy and Febrile Seizures Causes Retention of {alpha}1{beta}2{gamma}2S Receptors in the Endoplasmic Reticulum
J. Neurosci., October 6, 2004; 24(40): 8672 - 8677.
[Abstract] [Full Text] [PDF]

P. J. Groot-Kormelink, S. D. Broadbent, J. P. Boorman, and L. G. Sivilotti
Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
J. Gen. Physiol., June 1, 2004; 123(6): 697 - 708.
[Abstract] [Full Text] [PDF]

B. C. Drafts and J. L. Fisher
Structural Determinants of the Pharmacological Properties of the GABAA Receptor {alpha}6 Subunit
J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1108 - 1115.
[Abstract] [Full Text] [PDF]

F. Minier and E. Sigel
Positioning of the {alpha}-subunit isoforms confers a functional signature to {gamma}-aminobutyric acid type A receptors
PNAS, May 18, 2004; 101(20): 7769 - 7774.
[Abstract] [Full Text] [PDF]

D. Rusch, H. Zhong, and S. A. Forman
Gating Allosterism at a Single Class of Etomidate Sites on {alpha}1{beta}2{gamma}2L GABAA Receptors Accounts for Both Direct Activation and Agonist Modulation
J. Biol. Chem., May 14, 2004; 279(20): 20982 - 20992.
[Abstract] [Full Text] [PDF]

G. Akk, J. Bracamontes, and J. H. Steinbach
Activation of GABAA receptors containing the {alpha}4 subunit by GABA and pentobarbital
J. Physiol., April 15, 2004; 556(2): 387 - 399.
[Abstract] [Full Text] [PDF]

E. N. Goren, D. C. Reeves, and M. H. Akabas
Loose Protein Packing around the Extracellular Half of the GABAA Receptor {beta}1 Subunit M2 Channel-lining Segment
J. Biol. Chem., March 19, 2004; 279(12): 11198 - 11205.
[Abstract] [Full Text] [PDF]

D. Berezhnoy, Y. Nyfeler, A. Gonthier, H. Schwob, M. Goeldner, and E. Sigel
On the Benzodiazepine Binding Pocket in GABAA Receptors
J. Biol. Chem., January 30, 2004; 279(5): 3160 - 3168.
[Abstract] [Full Text] [PDF]

S. W. Baumann, R. Baur, and E. Sigel
Individual Properties of the Two Functional Agonist Sites in GABAA Receptors
J. Neurosci., December 3, 2003; 23(35): 11158 - 11166.
[Abstract] [Full Text] [PDF]

J. E. Coyle and D. B. Nikolov
GABARAP: Lessons for Synaptogenesis
Neuroscientist, June 1, 2003; 9(3): 205 - 216.
[Abstract] [PDF]

K. Bollan, D. King, L. A. Robertson, K. Brown, P. M. Taylor, S. J. Moss, and C. N. Connolly
GABAA Receptor Composition Is Determined by Distinct Assembly Signals within alpha and beta Subunits
J. Biol. Chem., February 7, 2003; 278(7): 4747 - 4755.
[Abstract] [Full Text] [PDF]

D. E. Raines, R. J. Claycomb, and S. A. Forman
Modulation of GABAA Receptor Function by Nonhalogenated Alkane Anesthetics: The Effects on Agonist Enhancement, Direct Activation, and Inhibition
Anesth. Analg., January 1, 2003; 96(1): 112 - 118.
[Abstract] [Full Text] [PDF]

G. W. Sawyer, D. C. Chiara, R. W. Olsen, and J. B. Cohen
Identification of the Bovine gamma -Aminobutyric Acid Type A Receptor alpha Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513
J. Biol. Chem., December 13, 2002; 277(51): 50036 - 50045.
[Abstract] [Full Text] [PDF]

P. A. Goldstein, F. P. Elsen, S.-W. Ying, C. Ferguson, G. E. Homanics, and N. L. Harrison
Prolongation of Hippocampal Miniature Inhibitory Postsynaptic Currents in Mice Lacking the GABAA Receptor alpha 1 Subunit
J Neurophysiol, December 1, 2002; 88(6): 3208 - 3217.
[Abstract] [Full Text] [PDF]

S. W. Baumann, R. Baur, and E. Sigel
Forced Subunit Assembly in alpha 1beta 2gamma 2 GABAA Receptors. INSIGHT INTO THE ABSOLUTE ARRANGEMENT
J. Biol. Chem., November 22, 2002; 277(48): 46020 - 46025.
[Abstract] [Full Text] [PDF]

E. Palma, V. Esposito, A. M. Mileo, G. Di Gennaro, P. Quarato, F. Giangaspero, C. Scoppetta, P. Onorati, F. Trettel, R. Miledi, et al.
Expression of human epileptic temporal lobe neurotransmitter receptors in Xenopus oocytes: An innovative approach to study epilepsy
PNAS, November 12, 2002; 99(23): 15078 - 15083.
[Abstract] [Full Text] [PDF]

M. Scheller and S. A. Forman
Coupled and Uncoupled Gating and Desensitization Effects by Pore Domain Mutations in GABAA Receptors
J. Neurosci., October 1, 2002; 22(19): 8411 - 8421.
[Abstract] [Full Text] [PDF]

I. Sarto, T. Klausberger, N. Ehya, B. Mayer, K. Fuchs, and W. Sieghart
A Novel Site on gamma 3 Subunits Important for Assembly of GABAA Receptors
J. Biol. Chem., August 16, 2002; 277(34): 30656 - 30664.
[Abstract] [Full Text] [PDF]

M. T. Bianchi, L. Song, H. Zhang, and R. L. Macdonald
Two Different Mechanisms of Disinhibition Produced by GABAA Receptor Mutations Linked to Epilepsy in Humans
J. Neurosci., July 1, 2002; 22(13): 5321 - 5327.
[Abstract] [Full Text] [PDF]

J. G. Newell and S. M. J. Dunn
Functional Consequences of the Loss of High Affinity Agonist Binding to gamma -Aminobutyric Acid Type A Receptors. IMPLICATIONS FOR RECEPTOR DESENSITIZATION
J. Biol. Chem., June 7, 2002; 277(24): 21423 - 21430.
[Abstract] [Full Text] [PDF]

J. H. Holden and C. Czajkowski
Different Residues in the GABAA Receptor alpha 1T60-alpha 1K70 Region Mediate GABA and SR-95531 Actions
J. Biol. Chem., May 17, 2002; 277(21): 18785 - 18792.
[Abstract] [Full Text] [PDF]

T. J. Jentsch, V. Stein, F. Weinreich, and A. A. Zdebik
Molecular Structure and Physiological Function of Chloride Channels
Physiol Rev, April 1, 2002; 82(2): 503 - 568.
[Abstract] [Full Text] [PDF]

S.-A. Thompson, P. B. Wingrove, L. Connelly, P. J. Whiting, and K. A. Wafford
Tracazolate Reveals a Novel Type of Allosteric Interaction with Recombinant gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., April 1, 2002; 61(4): 861 - 869.
[Abstract] [Full Text] [PDF]

S. B. Christie, C. P. Miralles, and A. L. De Blas
GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAA Receptor Clustering in Cultured Hippocampal Neurons
J. Neurosci., February 1, 2002; 22(3): 684 - 697.
[Abstract] [Full Text] [PDF]

P. A Davies, E. F Kirkness, and T. G Hales
Evidence for the formation of functionally distinct {alpha}{beta}{gamma}{varepsilon} GABAA receptors
J. Physiol., November 15, 2001; 537(1): 101 - 113.
[Abstract] [Full Text] [PDF]

C. E. Adkins, G. V. Pillai, J. Kerby, T. P. Bonnert, C. Haldon, R. M. McKernan, J. E. Gonzalez, K. Oades, P. J. Whiting, and P. B. Simpson
alpha 4beta 3delta GABAA Receptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential
J. Biol. Chem., October 12, 2001; 276(42): 38934 - 38939.
[Abstract] [Full Text] [PDF]

G. Hapfelmeier, R. Haseneder, E. Kochs, M. Beyerle, and W. Zieglgansberger
Coadministered Nitrous Oxide Enhances the Effect of Isoflurane on GABAergic Transmission by an Increase in Open-Channel Block
J. Pharmacol. Exp. Ther., July 1, 2001; 298(1): 201 - 208.
[Abstract] [Full Text]

A. J. Smith, L. Alder, J. Silk, C. Adkins, A. E. Fletcher, T. Scales, J. Kerby, G. Marshall, K. A. Wafford, R. M. McKernan, et al.
Effect of alpha Subunit on Allosteric Modulation of Ion Channel Function in Stably Expressed Human Recombinant gamma -Aminobutyric AcidA Receptors Determined Using 36Cl Ion Flux
Mol. Pharmacol., April 16, 2001; 59(5): 1108 - 1118.
[Abstract] [Full Text]

M. I. Strakhova, S. C. Harvey, C. M. Cook, J. M. Cook, and P. Skolnick
A Single Amino Acid Residue on the alpha 5 Subunit (Ile215) Is Essential for Ligand Selectivity at alpha 5beta 3gamma 2gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., April 13, 2001; 58(6): 1434 - 1440.
[Abstract] [Full Text]

M. T. Bianchi, K. F. Haas, and R. L. Macdonald
Structural Determinants of Fast Desensitization and Desensitization-Deactivation Coupling in GABAA Receptors
J. Neurosci., February 15, 2001; 21(4): 1127 - 1136.
[Abstract] [Full Text] [PDF]

J. P B Boorman, P. J GrootKormelink, and L. G Sivilotti
Stoichiometry of human recombinant neuronal nicotinic receptors containing the {beta}3 subunit expressed in Xenopus oocytes
J. Physiol., December 15, 2000; 529(3): 565 - 577.
[Abstract] [Full Text] [PDF]

G. Hapfelmeier, W. Zieglgansberger, R. Haseneder, H. Schneck, and E. Kochs
Nitrous Oxide and Xenon Increase the Efficacy of GABA at Recombinant Mammalian GABAA Receptors
Anesth. Analg., December 1, 2000; 91(6): 1542 - 1549.
[Abstract] [Full Text] [PDF]

A. S. Cohen, D. D. Lin, and D. A. Coulter
Protracted Postnatal Development of Inhibitory Synaptic Transmission in Rat Hippocampal Area CA1 Neurons
J Neurophysiol, November 1, 2000; 84(5): 2465 - 2476.
[Abstract] [Full Text] [PDF]

D. B. Williams and M. H. Akabas
Benzodiazepines Induce a Conformational Change in the Region of the gamma -Aminobutyric Acid Type A Receptor alpha 1-Subunit M3 Membrane-Spanning Segment
Mol. Pharmacol., November 1, 2000; 58(5): 1129 - 1136.
[Abstract] [Full Text]

J. G. Newell, M. Davies, A. N. Bateson, and S. M. J. Dunn
Tyrosine 62 of the gamma -Aminobutyric Acid Type A Receptor beta 2 Subunit Is an Important Determinant of High Affinity Agonist Binding
J. Biol. Chem., May 5, 2000; 275(19): 14198 - 14204.
[Abstract] [Full Text] [PDF]

J. E. Dalziel, G. B. Cox, P. W. Gage, and B. Birnir
Mutating the Highly Conserved Second Membrane-Spanning Region 9' Leucine Residue in the alpha 1 or beta 1 Subunit Produces Subunit-Specific Changes in the Function of Human alpha 1beta 1 gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., May 1, 2000; 57(5): 875 - 882.
[Abstract] [Full Text]

S. Neumahr, G. Hapfelmeier, M. Scheller, H. Schneck, C. Franke, and E. Kochs
Dual Action of Isoflurane on the {gamma}-aminobutyric Acid (GABA)-Mediated Currents Through Recombinant {alpha}1{beta}2{gamma}2L-GABAA-Receptor Channels
Anesth. Analg., May 1, 2000; 90(5): 1184 - 1190.
[Abstract] [Full Text] [PDF]

T. Klausberger, K. Fuchs, B. Mayer, N. Ehya, and W. Sieghart
GABAA Receptor Assembly. IDENTIFICATION AND STRUCTURE OF gamma 2 SEQUENCES FORMING THE INTERSUBUNIT CONTACTS WITH alpha 1 AND beta 3 SUBUNITS
J. Biol. Chem., March 17, 2000; 275(12): 8921 - 8928.
[Abstract] [Full Text] [PDF]

B. X. Carlson, A. C. Engblom, U. Kristiansen, A. Schousboe, and R. W. Olsen
A Single Glycine Residue at the Entrance to the First Membrane-Spanning Domain of the gamma -Aminobutyric Acid Type A Receptor beta 2 Subunit Affects Allosteric Sensitivity to GABA and Anesthetics
Mol. Pharmacol., March 1, 2000; 57(3): 474 - 484.
[Abstract] [Full Text]

S. Srinivasan, C. J. Nichols, G. M. Lawless, R. W. Olsen, and A. J. Tobin
Two Invariant Tryptophans on the alpha 1 Subunit Define Domains Necessary for GABAA Receptor Assembly
J. Biol. Chem., September 17, 1999; 274(38): 26633 - 26638.
[Abstract] [Full Text] [PDF]

T. R. Neelands and R. L. Macdonald
Incorporation of the pi Subunit into Functional gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., September 1, 1999; 56(3): 598 - 610.
[Abstract] [Full Text]

T. P. Bonnert, R. M. McKernan, S. Farrar, B. le Bourdelles, R. P. Heavens, D. W. Smith, L. Hewson, M. R. Rigby, D. J. S. Sirinathsinghji, N. Brown, et al.
theta , a novel gamma -aminobutyric acid type A receptor subunit
PNAS, August 17, 1999; 96(17): 9891 - 9896.
[Abstract] [Full Text] [PDF]

T. R. Neelands, J. Zhang, and R. L. Macdonald
GABAA Receptors Expressed in Undifferentiated Human Teratocarcinoma NT2 Cells Differ from Those Expressed by Differentiated NT2-N Cells
J. Neurosci., August 15, 1999; 19(16): 7057 - 7065.
[Abstract] [Full Text] [PDF]

C. Sur, S. J. Farrar, J. Kerby, P. J. Whiting, J. R. Atack, and R. M. McKernan
Preferential Coassembly of alpha 4 and delta �Subunits of the gamma -Aminobutyric AcidA Receptor in Rat Thalamus
Mol. Pharmacol., July 1, 1999; 56(1): 110 - 115.
[Abstract] [Full Text]

S. J. Farrar, P. J. Whiting, T. P. Bonnert, and R. M. McKernan
Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer
J. Biol. Chem., April 9, 1999; 274(15): 10100 - 10104.
[Abstract] [Full Text] [PDF]

S. Ueno, J. R. Trudell, E. I Eger II, and R. A. Harris
Actions of Fluorinated Alkanols on GABAA Receptors: Relevance to Theories of Narcosis
Anesth. Analg., April 1, 1999; 88(4): 877 - 877.
[Abstract] [Full Text] [PDF]

A. M L McClellan and R. E Twyman
Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors
J. Physiol., March 15, 1999; 515(3): 711 - 727.
[Abstract] [Full Text] [PDF]

P. Poisbeau, M. C. Cheney, M. D. Browning, and I. Mody
Modulation of Synaptic GABAA Receptor Function by PKA and PKC in Adult Hippocampal Neurons
J. Neurosci., January 15, 1999; 19(2): 674 - 683.
[Abstract] [Full Text] [PDF]

T. R. Neelands, J. L. Fisher, M. Bianchi, and R. L. Macdonald
Spontaneous and gamma -Aminobutyric Acid (GABA)-Activated GABAA Receptor Channels Formed by epsilon �Subunit-Containing Isoforms
Mol. Pharmacol., January 1, 1999; 55(1): 168 - 178.
[Abstract] [Full Text]

J. X. Connor, A. J. Boileau, and C. Czajkowski
A GABAA Receptor alpha 1 Subunit Tagged with Green Fluorescent Protein Requires a beta �Subunit for Functional Surface Expression
J. Biol. Chem., October 30, 1998; 273(44): 28906 - 28911.
[Abstract] [Full Text] [PDF]

T. Defazio and J. J. Hablitz
Zinc and Zolpidem Modulate mIPSCs in Rat Neocortical Pyramidal Neurons
J Neurophysiol, October 1, 1998; 80(4): 1670 - 1677.
[Abstract] [Full Text] [PDF]

R. M. McKernan, S. Farrar, I. Collins, F. Emms, A. Asuni, K. Quirk, and H. Broughton
Photoaffinity Labeling of the Benzodiazepine Binding Site of alpha 1beta 3gamma 2 gamma -Aminobutyric AcidA Receptors with Flunitrazepam Identifies a Subset of Ligands that Interact Directly with His102 of the alpha �Subunit and Predicts Orientation of These within the Benzodiazepine Pharmacophore
Mol. Pharmacol., July 1, 1998; 54(1): 33 - 43.
[Abstract] [Full Text]

P. J. Groot-Kormelink, W. H.�M.�L. Luyten, D. Colquhoun, and L. G. Sivilotti
A Reporter Mutation Approach Shows Incorporation of the "Orphan" Subunit beta 3 into a Functional Nicotinic Receptor
J. Biol. Chem., June 19, 1998; 273(25): 15317 - 15320.
[Abstract] [Full Text] [PDF]

E. A. Barnard, P. Skolnick, R. W. Olsen, H. Mohler, W. Sieghart, G. Biggio, C. Braestrup, A. N. Bateson, and S. Z. Langer
International Union of Pharmacology. XV. Subtypes of gamma -Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Structure and Receptor Function
Pharmacol. Rev., June 1, 1998; 50(2): 291 - 314.
[Abstract] [Full Text] [PDF]

J. Horenstein and M. H. Akabas
Location of a High Affinity Zn2+ Binding Site in the Channel of alpha 1beta 1 gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., May 1, 1998; 53(5): 870 - 877.
[Abstract] [Full Text]

M. Jechlinger, R. Pelz, V. Tretter, T. Klausberger, and W. Sieghart
Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABAA Receptors Containing alpha 6 Subunits
J. Neurosci., April 1, 1998; 18(7): 2449 - 2457.
[Abstract] [Full Text] [PDF]

M. D. Krasowski, S. E. Finn, Q. Ye, and N. L. Harrison
Trichloroethanol Modulation of Recombinant GABAA, Glycine and GABA rho 1 Receptors
J. Pharmacol. Exp. Ther., March 1, 1998; 284(3): 934 - 942.
[Abstract] [Full Text]

Y. Chang and D. S. Weiss
Substitutions of the Highly Conserved M2 Leucine Create Spontaneously Opening rho 1 gamma -Aminobutyric Acid Receptors
Mol. Pharmacol., March 1, 1998; 53(3): 511 - 523.
[Abstract] [Full Text]

M. D. Krasowski, V. V. Koltchine, C. E. Rick, Q. Ye, S. E. Finn, and N. L. Harrison
Propofol and Other Intravenous Anesthetics Have Sites of Action on the gamma -Aminobutyric Acid Type A Receptor Distinct from That for Isoflurane
Mol. Pharmacol., March 1, 1998; 53(3): 530 - 538.
[Abstract] [Full Text]

B. Ebert, S. A. Thompson, K. Saounatsou, R. McKernan, P. Krogsgaard-Larsen, and K. A. Wafford
Differences in Agonist/Antagonist Binding Affinity and Receptor Transduction Using Recombinant Human gamma -Aminobutyric Acid Type A Receptors
Mol. Pharmacol., December 1, 1997; 52(6): 1150 - 1156.
[Abstract] [Full Text]

P. Skolnick, R. J. Hu, C. M. Cook, S. D. Hurt, J. D. Trometer, R. Liu, Q. Huang, and J. M. Cook
[3H]RY 80: A High-Affinity, Selective Ligand for gamma -Aminobutyric AcidA Receptors Containing Alpha-5 Subunits,
J. Pharmacol. Exp. Ther., November 1, 1997; 283(2): 488 - 493.
[Abstract] [Full Text]

L. S. Premkumar and A. Auerbach
Stoichiometry of Recombinant N-Methyl-D-Aspartate Receptor Channels Inferred from Single-channel Current Patterns
J. Gen. Physiol., November 1, 1997; 110(5): 485 - 502.
[Abstract] [Full Text] [PDF]

P. B. Wingrove, S. A. Thompson, K. A. Wafford, and P. J. Whiting
Key Amino Acids in the gamma �Subunit of the gamma -Aminobutyric AcidA Receptor that Determine Ligand Binding and Modulation at the Benzodiazepine Site
Mol. Pharmacol., November 1, 1997; 52(5): 874 - 881.
[Abstract] [Full Text]

D. A. Coulter
Antiepileptic Drug Cellular Mechanisms of Action: Where Does Lamotrigine Fit In?
J Child Neurol, November 1, 1997; 12(1_suppl): S2 - S9.
[Abstract] [PDF]

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