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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 GABAA 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 GABAA 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 GABAA 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 1beta 2gamma 2 GABAA 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 GABAA 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 GABAA receptors must contain alpha , beta , and gamma  subunits to reconstitute the major features of native GABAA 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 GABAA 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 GABAA 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 1beta 2gamma 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 1beta 2gamma 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 mbeta 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 mbeta 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.
[View Larger Version of this Image (13K GIF file)]

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 MOmega . 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:
I=<LIM><OP>∑</OP><LL>j=1</LL><UL>x</UL></LIM> <FR><NU>I<SUB>max<SUB><IT>j</IT></SUB></SUB></NU><DE>1+<FENCE>EC<SUB>50<SUB>j</SUB></SUB>/[A]</FENCE><SUP>n<SUB>j</SUB></SUP></DE></FR>,
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), Imax 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 1beta 2gamma 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 mbeta gamma , alpha beta mgamma , 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 mbeta gamma , 0.30 ± 0.13 µM; alpha beta mgamma , 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 GABAA receptor. A, GABA-activated currents from oocytes expressing alpha beta gamma , alpha mbeta gamma , alpha beta mgamma , 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 mbeta gamma : 0.30 ± 0.050 µM, 0.85 ± 0.10 (n = 9); alpha beta mgamma : 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).
[View Larger Version of this Image (19K GIF file)]

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 GABAA 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 GABAA 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 mbeta 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 mbeta 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 mbeta 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 mbeta 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 mbeta 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 mgamma and alpha beta gamma gamma m coexpression to determine the number of beta  and gamma  subunits, respectively.
[View Larger Version of this Image (18K GIF file)]

alpha alpha mbeta 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 mbeta 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 mbeta 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 mbeta gamma receptors, and no intermediate component (attributable to alpha alpha mbeta 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 mbeta gamma , alpha beta beta mgamma , 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 GABAA receptor complex. The inset is a GABA dose-response relationship from an oocyte expressing primarily alpha beta gamma and alpha mbeta gamma receptors. This was achieved by injecting alpha beta gamma cRNA, waiting 5 d, and then injecting alpha mbeta 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 GABAA 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 GABAA receptor complex.
[View Larger Version of this Image (19K GIF file)]

alpha beta beta mgamma  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 mgamma 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 mgamma 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 GABAA 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 mbeta gamma and alpha beta beta mgamma 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 mbeta gamma and alpha beta beta mgamma 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 mbeta gamma coexpression. The dashed lines represent the mean EC50 determined from expression of alpha beta gamma and alpha mbeta 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 mgamma 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 mbeta gamma , alpha beta beta mgamma , 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 mbeta 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 mbeta 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 mgamma 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 mgamma 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 M) Fraction EC50M) Fraction EC50M) 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 mbeta gamma and alpha beta beta mgamma coexpression and two receptor types with alpha beta gamma gamma m coexpression, further supporting a pentameric GABAA 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 GABAA 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 1beta 2gamma 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 1beta 2gamma 2 GABAA 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 mbeta gamma , alpha beta beta mgamma , 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 mbeta gamma and alpha beta beta mgamma 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 mbeta gamma and alpha beta mgamma 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 mbeta gamma and alpha beta beta mgamma 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 6beta 2gamma 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 1beta 2gamma 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 GABAA 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 3beta 2gamma 2 receptors in transfected human embryonic kidney cells (HEK293) and concluded that the possible subunit stoichiometries for this receptor were 2alpha 1beta 2gamma , 2alpha 2beta 1gamma , or 1alpha 2beta 2gamma , of which 2alpha 1beta 2gamma 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 GABAA 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 GABAA 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 GABAA 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.



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M. D. Krasowski, S. E. Finn, Q. Ye, and N. L. Harrison
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Y. Chang and D. S. Weiss
Substitutions of the Highly Conserved M2 Leucine Create Spontaneously Opening rho 1 gamma -Aminobutyric Acid Receptors
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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
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P. Skolnick, R. J. Hu, C. M. Cook, S. D. Hurt, J. D. Trometer, R. Liu, Q. Huang, and J. M. Cook
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Antiepileptic Drug Cellular Mechanisms of Action: Where Does Lamotrigine Fit In?
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G. H. Gorrie, Y. Vallis, A. Stephenson, J. Whitfield, B. Browning, T. G. Smart, and S. J. Moss
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S. M. O'Shea and N. L. Harrison
Arg-274 and Leu-277 of the gamma -Aminobutyric Acid Type A Receptor alpha 2 Subunit Define Agonist Efficacy and Potency
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