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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

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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 , , and  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 1(L263S), 2(L259S), and 2(L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g.,  and L263S), along with their wild-type counterparts (e.g.,  and ), 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 (, , and ) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant 122 GABA_A receptor is a pentamer composed of two  subunits, two  subunits, and one  subunit.

Key words: GABA; stoichiometry; receptor; ion channel; mutagenesis

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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: , , , and  (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 , , and  subunit classes. Exogenous expression of various combinations of subunits suggests that GABA_A receptors must contain , , and  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 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 122 GABA receptors expressed in Xenopus oocytes. Mutation of this leucine to serine in either the rat 1, 2, or 2 GABA subunit isoforms increased the sensitivity to GABA. Coexpression of wild-type and mutant subunits of each class (e.g.,  and -mutant), along with their wild-type counterparts (e.g.,  and ), 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 122 GABA receptor is a pentamer composed of two  subunits, two  subunits, and one  subunit.

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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 1(L263S): 5-CAA GGT TGT CAT GGT ACT AAC GGT CGT CAC TCC-3; 2(L259S): 5-GAT TGT GGT CAT CGT ACT GAC AGT TGT AAT TCC-3; and 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 :: 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).

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Fig. 5. The stoichiometry does not depend on the cRNA injection ratio. The _m and _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 _m 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 _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  and  subunits in the GABA receptor is fixed.
[View Larger Version of this Image (13K GIF file)]

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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. 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.

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RESULTS

Mutation of the conserved leucine in 1, 2, or 2 increases the GABA sensitivity

The isoforms 1, 2, and 2 are widely distributed in the brain (Benke et al., 1991; Wisden et al., 1992; Ruano et al., 1994), so we selected the 122 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 , , and  isoforms. In this manuscript, we designate the rat 1, 2, and 2 subunit isoforms as simply , , and .

A conserved leucine in the putative second membrane-spanning domain (TM2) was mutated to serine in the  (L263S),  (L259S), and  (L274S) subunits. (Henceforth, the subunits with the leucine mutation will be designated _m, _m, and _m). cRNA was in vitro transcribed for both mutant and wild-type , , and  subunits, mixed in the combinations , _m, _m, and _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 _m, _m, or _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) , 45.8 ± 3.6 µM; _m, 0.30 ± 0.13 µM; _m, 0.035 ± 0.004 µM; and _m, 0.99 ± 0.23 µM.

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Fig. 1. Mutation of the conserved leucine in TM2 of the 1(L263S), 2(L259S), and 2(L274S) subunit increases the GABA sensitivity of the GABA_A receptor. A, GABA-activated currents from oocytes expressing , _m, _m, and _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 : 45.8 ± 3.6 µM, 1.59 ± 0.09 (n = 5); _m: 0.30 ± 0.050 µM, 0.85 ± 0.10 (n = 9); _m: 0.035 ± 0.004 µM, 1.12 ± 0.04 (n = 3); and _m: 0.99 ± 0.23 µM, 1.78 ± 0.36 (n = 4).
[View Larger Version of this Image (19K GIF file)]

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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 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 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 _m, _m, and _m, respectively. Because the effects of the mutations may not be equivalent for , , and  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 (, , , or ) 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 _m, _m, and _m.

Coexpression of wild-type and mutant subunits

Predictions Assume there is only one  subunit in the GABA_A receptor complex. Coexpression of both  and _m subunits along with wild-type  and  subunits would result in GABA dose-response relationships having two components (Fig. 2A): one component from activation of the _m receptors and one component from activation of the receptors. Furthermore, on the basis of the data presented in Figure 1, the GABA EC₅₀ values of these two components would be 0.30 and 46 µM, respectively. Alternatively, if the GABA receptor complex contained two  subunits, the GABA dose-response relationships resulting from coexpression of _m, , , and  subunits would have three components (Fig. 2B): one component from receptors in which both  subunits are wild type (EC₅₀ 46 µM), one component from receptors in which both  subunits are mutant (EC₅₀ 0.30 µM), and one component with an intermediate GABA sensitivity from receptors with one wild-type and one mutant  subunit. If mutation of the two  subunits has an equivalent effect, the shift in sensitivity contributed by each _m subunit would be the square root of the shift in EC₅₀ observed when both  subunits are mutant (153-fold; Fig. 1B). Thus, each _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 _m coexpression, assuming three  subunits in the receptor complex.)

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Fig. 2. The number of components comprising the GABA dose-response relationship with _m coexpression depends on the number of  subunits in the GABA receptor complex. A, With one  subunit in the GABA receptor complex, the GABA dose-response relationship from oocytes coexpressing , _m, , and  subunits would be composed of two components (continuous line): one component from activation of receptors and one component from activation of _m receptors (dashed lines, scaled to facilitate comparison with the continuous line). The EC₅₀ values of these two components would be 0.30 and 45.8 µM (indicated on the abscissa) as demonstrated by the data in Figure 1. The mutant  subunit is shown shaded. B, With two  subunits in the GABA receptor complex, the GABA dose-response relationship from oocytes coexpressing , _m, , and  subunits would be composed of three components (continuous line): one component from activation of receptors, one component from activation of _m receptors, and an intermediate component from activation of receptors containing both an  and an _m subunit (dashed lines). The EC₅₀ values of the first and third components would be 0.30 and 45.8 µM, and the predicted EC₅₀ of the intermediate component, assuming the two _m subunits contribute equally to the shift, would be 3.7 µM. A similar logic could be applied to _m and _m coexpression to determine the number of  and  subunits, respectively.
[View Larger Version of this Image (18K GIF file)]

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_m coexpression Figure 3A shows the results from experiments in which _m, , , and  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 _m coexpression (0.30 ± 0.13 µM), and the EC₅₀ of the third component, 36.3 ± 8.1 µM, corresponds to that of the 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  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  and an _m subunit. This interpretation is supported by the experiment shown in the inset of Figure 3A. Oocytes were injected with cRNA; 5 d later, the same oocytes were injected with _m 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 and _m receptors, and no intermediate component (attributable to _m 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).

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Fig. 3. GABA dose-response relationships from coexpression of _m, _m, and _m subunits. A, cRNA encoding for , _m, , and  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 (-to-_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  subunits in the GABA_A receptor complex. The inset is a GABA dose-response relationship from an oocyte expressing primarily and _m receptors. This was achieved by injecting cRNA, waiting 5 d, and then injecting _m cRNA. This ensures that  and _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 , , _m, and  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 (-to-_m cRNA injection ratio = 1:1). The three components suggest that there are two  subunits in the GABA_A receptor complex. C, cRNA encoding for , , , and _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 (-to-_m cRNA injection ratio = 1:1). The dashed line represents the first component. Two components suggest that there is one  subunit in the GABA_A receptor complex.
[View Larger Version of this Image (19K GIF file)]

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_m coexpression Figure 3B presents the average GABA dose-response relationship from oocytes coexpressing  and _m subunits along with wild-type  and  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 _m coexpression (0.035 ± 0.004 µM), and the EC₅₀ of the third component, 39.2 ± 7.9 µM, corresponds to that of the 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  subunit (Fig. 2B). Assuming two  subunits and the shift observed in Figure 1B for _m coexpression (1308-fold), each _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. _m coexpression The data presented thus far (Fig. 3A,B) suggest the presence of two  subunits and two  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  subunit. This inferred presence of one  subunit is confirmed by the _m coexpression studies presented in Figure 3C. In contrast to that of _m and _m coexpression, the _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 _m coexpression (0.99 ± 0.23), and the EC₅₀ of the second component, 40.9 ± 4.8 µM, corresponds to that determined for receptors (45.8 ± 3.6 µM). Therefore, the two components in Figure 3C represent the activation of receptors containing either a  or _m subunit, and the GABA receptor complex contains only one  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.,  and _m) coinjected with their wild-type counterparts (e.g.,  and ) 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 -to-_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 -to-_m and -to-_m cRNA ratio.

At all cRNA injection ratios tested, the GABA dose-response relationships consisted of three components for _m and _m coexpression and two components for _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 -to-_m cRNA. Figure 4A is a plot of the EC₅₀ values of the three components versus the fraction of the wild-type component for _m coexpression. The dashed lines represent the mean EC₅₀ determined from expression of and _m (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  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  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 _m and _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.

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Fig. 4. The EC₅₀ values of the components with _m, _m, and _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 _m 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 -to-_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  and _m cRNA remained fixed with respect to  and . For all -to-_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 and _m 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  subunits in the GABA receptor complex. B, Similar plot as in A, but for _m coexpression at different ratios of  to _m (1:1 and 3:1). The dashed lines represent the mean EC₅₀ values determined from and _m coexpression (Fig. 1). These data indicate three components and hence two  subunits in the GABA receptor complex. C, Similar plot as in A, but for _m coexpression at different ratios of  to _m (1:3 and 1:1). These data indicate two components and hence one  subunit in the GABA receptor complex.
[View Larger Version of this Image (18K GIF file)]

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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  m(1:3) 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  m(1:1) 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  m(3:1) 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  m(1:1) 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  m(3:1) 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  m(1:3) 1.16  ± 0.14 0.58  ± 0.12 47.9  ± 19.8 0.42  ± 0.12 3  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 :: 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 _m and _m coexpression and two receptor types with _m coexpression, further supporting a pentameric GABA_A receptor composed of two  subunits, two  subunits, and one  subunit.

The stoichiometry does not depend on the relative availability of , , and  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 _m, , and  cRNA at a ratio of 4:1:1 or , , and _m cRNA at a ratio of 1:1:4. If the increased availability of  or  subunits can increase the number of  or  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  and  subunits in the GABA receptor is fixed. By default, the number of  subunits should be fixed as well.

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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 122 receptors are pentamers composed of two  subunits, two  subunits, and one  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 122 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., , _m) and coexpressed this mixture along with their wild-type counterparts (e.g., , ). 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., , , etc.) for the following reasons. (1) Expression of , , or  subunits alone, as well as / coexpression, does not yield functional GABA receptors in oocytes. (2) receptors have an EC₅₀ of 30.1 ± 8.4 µM, which is similar to that of GABA receptors (45.8 ± 3.6 µM). (3) 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 _m, _m, and _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 _m and _m coexpression and 0.50 for _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 _m and _m coexpression, we observed a picrotoxin-sensitive current in the absence of GABA that was not observed in oocytes expressing _m or 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 _m and _m 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  subunit. In addition, Im et al. (1995), using tandem constructs of 6-2 GABA subunits, concluded that 622 GABA receptors contain two  subunits, two  subunits, and one  subunit.

Our results are in contrast to those of Benke et al. (1994), which infer that only one  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 1, 2, or 3 antiserum sum to ~100% of the total binding. If neurons dominantly express one isoform of the  subunit, however, the results of Benke et al. (1994) would be obtained regardless of the number of  subunits in the GABA receptor complex. In fact, immunolocalization studies indicate some segregation of the  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 2 and 3 subunit antisera was slightly greater than the [³H]muscimol binding observed with 2 and 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 2S and 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  subunit. Furthermore, GABA receptors obtained by 3 immunoprecipitation were labeled in a Western blot probed with 2 antisera, again suggesting that 2 and 3 subunits can coexist in the same GABA receptor complex. This conclusion seems to contradict our findings that the 122 GABA receptor contains a single copy of the 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 or GABA receptors that may contain multiple  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 , , and  subunit isoforms.

Finally, Backus et al. (1993) examined the stoichiometry of rat 322 receptors in transfected human embryonic kidney cells (HEK293) and concluded that the possible subunit stoichiometries for this receptor were 212, 221, or 122, of which 212 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 3, 2, and 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  subunits, two  subunits, and one  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  subunit and two domains on the  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 - 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 - interface or at a - or - 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.

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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.

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REFERENCES

• Amin J, Weiss DS (1993) GABA_A receptor needs two homologous domains of the  subunit for activation by GABA, but not by pento