Skip to main content

Main menu

  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Current Issue
    • Issue Archive
    • Collections
    • Podcast
  • ALERTS
  • FOR AUTHORS
    • Information for Authors
    • Fees
    • Journal Clubs
    • eLetters
    • Submit
  • EDITORIAL BOARD
  • ABOUT
    • Overview
    • Advertise
    • For the Media
    • Rights and Permissions
    • Privacy Policy
    • Feedback
  • SUBSCRIBE

User menu

  • Log out
  • Log in
  • My Cart

Search

  • Advanced search
Journal of Neuroscience
  • Log out
  • Log in
  • My Cart
Journal of Neuroscience

Advanced Search

Submit a Manuscript
  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Current Issue
    • Issue Archive
    • Collections
    • Podcast
  • ALERTS
  • FOR AUTHORS
    • Information for Authors
    • Fees
    • Journal Clubs
    • eLetters
    • Submit
  • EDITORIAL BOARD
  • ABOUT
    • Overview
    • Advertise
    • For the Media
    • Rights and Permissions
    • Privacy Policy
    • Feedback
  • SUBSCRIBE
PreviousNext
Articles

Stoichiometry of a Recombinant GABAA Receptor

Yongchang Chang, Ruoping Wang, Sonal Barot and David S. Weiss
Journal of Neuroscience 1 September 1996, 16 (17) 5415-5424; DOI: https://doi.org/10.1523/JNEUROSCI.16-17-05415.1996
Yongchang Chang
1Neurobiology Research Center, Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ruoping Wang
1Neurobiology Research Center, Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sonal Barot
1Neurobiology Research Center, Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David S. Weiss
1Neurobiology Research Center, Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

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 α, β, and γ subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABAAreceptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat α1(αL263S), β2(αL259S), and γ2(αL274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., α and αL263S), along with their wild-type counterparts (e.g., β and γ), 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 (α, β, and γ) could then be determined from the number of components comprising the compound GABA dose–response relationships. Using this approach, we conclude that the recombinant α1β2γ2 GABAA receptor is a pentamer composed of two α subunits, two β subunits, and one γ subunit.

  • GABA
  • stoichiometry
  • receptor
  • ion channel
  • mutagenesis

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: α, β, γ, and δ (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 α, β, and γ subunit classes. Exogenous expression of various combinations of subunits suggests that GABAAreceptors must contain α, β, and γ 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 GABAAreceptor.) 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 α1β2γ2 GABA receptors expressed in Xenopus oocytes. Mutation of this leucine to serine in either the rat α1, β2, or γ2 GABA subunit isoforms increased the sensitivity to GABA. Coexpression of wild-type and mutant subunits of each class (e.g., α and α-mutant), along with their wild-type counterparts (e.g., β and γ), 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 α1β2γ2 GABA receptor is a pentamer composed of two α subunits, two β subunits, and one γ subunit.

MATERIALS AND METHODS

Site-directed mutagenesis and in vitrotranscription. 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 α1(L263S): 5′-CAA GGT TGT CAT GGT ACT AAC GGT CGT CAC TCC-3′; β2(L259S): 5′-GAT TGT GGT CAT CGT ACT GAC AGT TGT AAT TCC-3′; and γ2(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 vitrotranscription 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 α:β:γ 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.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 5.

The stoichiometry does not depend on the αβγ cRNA injection ratio. The αmβγ and αβγm 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 EC50values of the GABA dose–response relationships for αmβγ 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 αβγm 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 α and γ subunits in the GABA receptor is fixed.

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 MΩ. 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: Embedded Imagewhere 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 α1, β2, or γ2 increases the GABA sensitivity

The isoforms α1, β2, and γ2 are widely distributed in the brain (Benke et al., 1991; Wisden et al., 1992; Ruano et al., 1994), so we selected the α1β2γ2 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 α, β, and γ isoforms. In this manuscript, we designate the rat α1, β2, and γ2 subunit isoforms as simply α, β, and γ.

A conserved leucine in the putative second membrane-spanning domain (TM2) was mutated to serine in the α (αL263S), β (βL259S), and γ (γL274S) subunits. (Henceforth, the subunits with the leucine mutation will be designated αm, βm, and γm). cRNA was in vitro transcribed for both mutant and wild-type α, β, and γ subunits, mixed in the combinations αβγ, αmβγ, αβmγ, and αβγm, and injected into Xenopus laevisoocytes. 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 αm, βm, or γm subunit. Figure 1Bshows 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) αβγ, 45.8 ± 3.6 μm; αmβγ, 0.30 ± 0.13 μm; αβmγ, 0.035 ± 0.004 μm; and αβγm, 0.99 ± 0.23 μm.

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Mutation of the conserved leucine in TM2 of the α1(L263S), β2(L259S), and γ2(L274S) subunit increases the GABA sensitivity of the GABAA receptor. A, GABA-activated currents from oocytes expressing αβγ, αmβγ, αβmγ, and αβγm 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 αβγ: 45.8 ± 3.6 μm, 1.59 ± 0.09 (n = 5); αmβγ: 0.30 ± 0.050 μm, 0.85 ± 0.10 (n = 9); αβmγ: 0.035 ± 0.004 μm, 1.12 ± 0.04 (n = 3); and αβγm: 0.99 ± 0.23 μm, 1.78 ± 0.36 (n = 4).

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 GABAAreceptor 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 αm, βm, and γm, respectively. Because the effects of the mutations may not be equivalent for α, β, and γ 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 (α, β, γ, or δ) 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 αm, βm, and γm.

Coexpression of wild-type and mutant subunits

Predictions

Assume there is only one α subunit in the GABAAreceptor complex. Coexpression of both α and αmsubunits along with wild-type β and γ subunits would result in GABA dose–response relationships having two components (Fig.2A): one component from activation of the αmβγ receptors and one component from activation of the αβγ 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 α subunits, the GABA dose–response relationships resulting from coexpression of αm, α, β, and γ subunits would have three components (Fig. 2B): one component from receptors in which both α subunits are wild type (EC50 ≈46 μm), one component from receptors in which both α subunits are mutant (EC50 ≈0.30 μm), and one component with an intermediate GABA sensitivity from receptors with one wild-type and one mutant α subunit. If mutation of the two α subunits has an equivalent effect, the shift in sensitivity contributed by each αm subunit would be the square root of the shift in EC50 observed when both α subunits are mutant (153-fold; Fig. 1B). Thus, each αm 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 ααmβγ coexpression, assuming three α subunits in the receptor complex.)

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

The number of components comprising the GABA dose–response relationship with ααmβγ coexpression depends on the number of α subunits in the GABA receptor complex.A, With one α subunit in the GABA receptor complex, the GABA dose–response relationship from oocytes coexpressing α, αm, β, and γ subunits would be composed of two components (continuous line): one component from activation of αβγ receptors and one component from activation of αmβγ 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 α subunit is shownshaded. B, With two α subunits in the GABA receptor complex, the GABA dose–response relationship from oocytes coexpressing α, αm, β, and γ subunits would be composed of three components (continuous line): one component from activation of αβγ receptors, one component from activation of αmβγ receptors, and an intermediate component from activation of receptors containing both an α and an αm 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 αm subunits contribute equally to the shift, would be 3.7 μm. A similar logic could be applied to αββmγ and αβγγm coexpression to determine the number of β and γ subunits, respectively.

ααmβγ coexpression

Figure 3A shows the results from experiments in which αm, α, β, and γ 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 αmβγ coexpression (0.30 ± 0.13 μm), and the EC50 of the third component, 36.3 ± 8.1 μm, corresponds to that of the αβγ 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 α 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 α and an αm subunit. This interpretation is supported by the experiment shown in the inset of Figure 3A. Oocytes were injected with αβγ cRNA; 5 d later, the same oocytes were injected with αmβγ cRNA. Previous studies demonstrate that the cRNA for glycine subunits (Kuhse et al., 1993) or GABA subunits (Amin and Weiss, 1996) injected into Xenopusoocytes is degraded within 2–3 d. Therefore, after the second injection, the oocytes should contain only αβγ and αmβγ receptors, and no intermediate component (attributable to ααmβγ 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 EC50values of (mean ± SD) 0.42 ± 0.17 μm and 30.0 ± 7.5 μm (n = 3).

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

GABA dose–response relationships from coexpression of ααmβγ, αββmγ, and αβγγm subunits. A, cRNA encoding for α, αm, β, and γ 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 (α-to-αm cRNA injection ratio = 1:3). The dashed linesrepresenting 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 α subunits in the GABAA receptor complex. The inset is a GABA dose–response relationship from an oocyte expressing primarily αβγ and αmβγ receptors. This was achieved by injecting αβγ cRNA, waiting 5 d, and then injecting αmβγ cRNA. This ensures that α and αmcRNAs 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 byarrows). 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 α, β, βm, and γ 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 (β-to-βm cRNA injection ratio = 1:1). The three components suggest that there are two β subunits in the GABAA receptor complex. C, cRNA encoding for α, β, γ, and γm 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(γ-to-γm cRNA injection ratio = 1:1). Thedashed line represents the first component. Two components suggest that there is one γ subunit in the GABAA receptor complex.

αββmγ coexpression

Figure 3B presents the average GABA dose–response relationship from oocytes coexpressing β and βm subunits along with wild-type α and γ 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 αβmγ coexpression (0.035 ± 0.004 μm), and the EC50 of the third component, 39.2 ± 7.9 μm, corresponds to that of the αβγ 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 β subunit (Fig. 2B). Assuming two β subunits and the shift observed in Figure 1B for αβmγ coexpression (1308-fold), each βm 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.

αβγγm coexpression

The data presented thus far (Fig. 3A,B) suggest the presence of two α subunits and two β 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 γ subunit. This inferred presence of one γ subunit is confirmed by the αβγγm coexpression studies presented in Figure 3C. In contrast to that of ααmβγ and αββmγ coexpression, the αβγγm 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 αβγm coexpression (0.99 ± 0.23), and the EC50 of the second component, 40.9 ± 4.8 μm, corresponds to that determined for αβγ receptors (45.8 ± 3.6 μm). Therefore, the two components in Figure 3C represent the activation of receptors containing either a γ or γm subunit, and the GABA receptor complex contains only one γ 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., α and αm) coinjected with their wild-type counterparts (e.g., β and γ) 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 α-to-αm 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 β-to-βm and γ-to-γm cRNA ratio.

At all cRNA injection ratios tested, the GABA dose–response relationships consisted of three components for ααmβγ and αββmγ coexpression and two components for αβγγm coexpression. Figure4 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 α-to-αm cRNA. Figure 4A is a plot of the EC50 values of the three components versus the fraction of the wild-type component for ααmβγ coexpression. The dashed lines represent the mean EC50determined from expression of αβγ and αmβγ (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 α 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 α 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. Figure4B,C are similar plots of the EC50 values as a function of the fraction of the wild-type component with αββmγ and αβγγm 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.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

The EC50 values of the components with ααmβγ, αββmγ, and αβγγm 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 ααmβγ 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 α-to-αm 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 α and αm cRNA remained fixed with respect to β and γ. For all α-to-αm ratios tested, the GABA dose–response relationships were described by the sum of three Hill equations. Thedashed lines represent the mean EC50 values determined from αβγ and αmβγ 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 α subunits in the GABA receptor complex. B, Similar plot as inA, but for αββmγ coexpression at different ratios of β to βm (1:1 and 3:1). Thedashed lines represent the mean EC50 values determined from αβγ and αβmγ coexpression (Fig.1). These data indicate three components and hence two β subunits in the GABA receptor complex. C, Similar plot as inA, but for αβγγm coexpression at different ratios of γ to γm (1:3 and 1:1). These data indicate two components and hence one γ subunit in the GABA receptor complex.

View this table:
  • View inline
  • View popup
Table 1.

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

Nevertheless, the data presented in Figure 4 suggest that in terms of GABA sensitivity, there are three receptor types with ααmβγ and αββmγ coexpression and two receptor types with αβγγm coexpression, further supporting a pentameric GABAA receptor composed of two α subunits, two β subunits, and one γ subunit.

The stoichiometry does not depend on the relative availability of α, β, and γ 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 αm, β, and γ cRNA at a ratio of 4:1:1 or α, β, and γm cRNA at a ratio of 1:1:4. If the increased availability of α or γ subunits can increase the number of α or γ subunits incorporated into the GABA receptor, the EC50values 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 α and γ subunits in the GABA receptor is fixed. By default, the number of β 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 α1β2γ2 receptors are pentamers composed of two α subunits, two β subunits, and one γ 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 α1β2γ2 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., α, αm) and coexpressed this mixture along with their wild-type counterparts (e.g., β, γ). Although the relative amplitudes of the components depended on the wild-type-to-mutant cRNA ratio, the observed number of components and their EC50values were essentially independent of the cRNA ratio. This supports the conclusion that the dose–response relationships in Figure3A–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., αβ, βγ, etc.) for the following reasons. (1) Expression of α, β, or γ subunits alone, as well as α/γ coexpression, does not yield functional GABA receptors in oocytes. (2) βγ receptors have an EC50 of 30.1 ± 8.4 μm, which is similar to that of αβγ GABA receptors (45.8 ± 3.6 μm). (3) αβ 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 ααmβγ, αββmγ, and αβγγm, 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 ααmβγ and αββmγ coexpression and 0.50 for αβγγm 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 αmβγ and αβmγ coexpression, we observed a picrotoxin-sensitive current in the absence of GABA that was not observed in oocytes expressing αβγm or αβγ 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 ααmβγ and αββmγ 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 and4.

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 α subunit. In addition, Im et al. (1995), using tandem constructs of α6-β2 GABA subunits, concluded that α6β2γ2 GABA receptors contain two α subunits, two β subunits, and one γ subunit.

Our results are in contrast to those of Benke et al. (1994), which infer that only one β 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 β1, β2, or β3 antiserum sum to ∼100% of the total binding. If neurons dominantly express one isoform of the β subunit, however, the results of Benke et al. (1994) would be obtained regardless of the number of β subunits in the GABA receptor complex. In fact, immunolocalization studies indicate some segregation of the β 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 γ2 and γ3 subunit antisera was slightly greater than the [3H]muscimol binding observed with γ2 and γ3 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 γ2S and γ2L 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 γ subunit. Furthermore, GABA receptors obtained by γ3 immunoprecipitation were labeled in a Western blot probed with γ2 antisera, again suggesting that γ2 and γ3 subunits can coexist in the same GABA receptor complex. This conclusion seems to contradict our findings that the α1β2γ2 GABA receptor contains a single copy of the γ2 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 GABAAreceptors) or (2) a subpopulation of βγ or αγ GABA receptors that may contain multiple γ 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 α, β, and γ subunit isoforms.

Finally, Backus et al. (1993) examined the stoichiometry of rat α3β2γ2 receptors in transfected human embryonic kidney cells (HEK293) and concluded that the possible subunit stoichiometries for this receptor were 2α1β2γ, 2α2β1γ, or 1α2β2γ, of which 2α1β2γ 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 α3, β2, and γ2 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 α subunits, two β subunits, and one γ 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 α subunit and two domains on the β 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 α–β 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 α-β interface or at a β-γ or α-γ 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

  • 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

  1. ↵
    1. Amin J,
    2. Weiss DS
    (1993) GABAA receptor needs two homologous domains of the β subunit for activation by GABA, but not by pentobarbital. Nature 366:565–569.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Amin J,
    2. Weiss DS
    (1996) Insight into the activation mechanism of ρ1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits. Proc R Soc Lond [Biol] 263:273–282.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Amin J,
    2. Dickerson IM,
    3. Weiss DS
    (1994) The agonist binding site of the GABAA channel is not formed by the extracellular cysteine loop. Mol Pharmacol 45:317–323.
    OpenUrlAbstract
  4. ↵
    1. Anand R,
    2. Conroy WG,
    3. Schoepfer R,
    4. Whiting P,
    5. Lindstrom J
    (1991) Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure. J Biol Chem 266:11192–11198.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Backus KH,
    2. Arigoni M,
    3. Drescher U,
    4. Scheurer L,
    5. Malherbe P,
    6. Mohler H,
    7. Benson JA
    (1993) Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification. NeuroReport 5:285–288.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Behe P,
    2. Stern P,
    3. Wyllie DJA,
    4. Nassar M,
    5. Schoepfer R,
    6. Colquhoun D
    (1995) Determination of NMDA NR1 subunit copy number in recombinant NMDA receptor. Proc R Soc Lond [Biol] 262:205–213.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Benke D,
    2. Mertens S,
    3. Trzeciak A,
    4. Gillessen D,
    5. Mohler H
    (1991) GABAA receptors display association of γ2-subunit with α1- and β2/3-subunits. J Biol Chem 266:4478–4483.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Benke D,
    2. Fritschy J-M,
    3. Trzeciak A,
    4. Bannwarth W,
    5. Mohler H
    (1994) Distribution, prevalence, and drug binding profile of γ-aminobutyric acid type A receptor subtypes differing in the β subunit variant. J Biol Chem 268:27100–27107.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Blount P,
    2. Merlie JP
    (1989) Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron 3:349–357.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Colquhoun D,
    2. Ogden DC
    (1988) Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J Physiol (Lond) 395:131–159.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Cooper E,
    2. Couturier S,
    3. Ballivet M
    (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature 350:235–238.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Czajkowski C,
    2. Karlin A
    (1991) Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor: a negatively charged region of the δ subunit within 0.9 nm of the alpha subunit binding site disulfide. J Biol Chem 266:22603–22612.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Duggan MJ,
    2. Pollard S,
    3. Stephenson FA
    (1991) Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers: demonstration of receptor populations containing α1, α2:α1α3, and α2α3 subunit pairs. J Biol Chem 266:24778–24784.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Filatov GN,
    2. White MM
    (1995) The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating. Mol Pharmacol 48:379–384.
    OpenUrlAbstract
  15. ↵
    1. Harvey RJ,
    2. Kim H-C,
    3. Darlison MG
    (1993) Molecular cloning reveals the existence of a fourth gamma subunit of the vertebrate brain GABAA receptor. FEBS Lett 331:211–216.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Im WB,
    2. Pregenzer JF,
    3. Binder JA,
    4. Dillon GH,
    5. Alberts GL
    (1995) Chloride channel expression with the tandem construct of α6-β2 GABAA receptor subunit requires a monomeric subunit of α6 or γ2. J Biol Chem 270:26063–26066.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Khan ZU,
    2. Gutierrez A,
    3. De Blas AL
    (1994) Short and long form γ2 subunits of the GABAA/benzodiazepine receptors. J Neurochem 63:1466–1476.
    OpenUrlPubMed
  18. ↵
    1. Khrestchatisky M,
    2. MacLennan AJ,
    3. Chiang MY,
    4. Xu WT,
    5. Jackson MB,
    6. Brecha N,
    7. Sternini C,
    8. Olsen RW,
    9. Tobin AJ
    (1989) A novel α subunit in rat brain GABA receptors. Neuron 3:745–753.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Kuhse J,
    2. Laube B,
    3. Magalei D,
    4. Betz H
    (1993) Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry. Neuron 11:1049–1056.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Kurosaki T,
    2. Fukuda K,
    3. Konno T,
    4. Mori Y,
    5. Tanaka K,
    6. Mishina M,
    7. Numa S
    (1987) Functional properties of nicotinic acetylcholine receptor subunits expressed in various combinations. FEBS Lett 214:253–258.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Labarca C,
    2. Nowak MW,
    3. Zhang H,
    4. Tang L,
    5. Deshpande P,
    6. Lester HA
    (1995) Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature 376:514–516.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Langosch D,
    2. Thomas L,
    3. 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.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Levitan ES,
    2. Blair LAC,
    3. Dionne VE,
    4. Barnard EA
    (1988) Biophysical and pharmacological properties of cloned GABAA receptor subunits expressed in Xenopus oocytes. Neuron 1:773–781.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Lindstrom J,
    2. Merlie J,
    3. Yogeeswaran G
    (1979) Biochemical properties of acetylcholine receptor subunits from Torpedo californica . Biochemistry 18:4465–4470.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Lolait SJ,
    2. O’Carroll AM,
    3. Kusano K,
    4. Muller JM,
    5. Brownstein MJ,
    6. Mahan LC
    (1989) Cloning and expression of a novel rat GABA receptor. FEBS Lett 246:145–148.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Luddens H,
    2. Killisch I,
    3. Seeburg PH
    (1991) More than one α-variant may exist in a GABA/benzodiazepine receptor complex. J Recept Res 11:535–551.
    OpenUrlPubMed
  27. ↵
    1. Macdonald RL,
    2. Olsen RW
    (1994) GABA receptor channels. Annu Rev Neurosci 17:569–602.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Malherbe P,
    2. Sigel E,
    3. Baur R,
    4. Persohn E,
    5. Richards J,
    6. Mohler H
    (1990) Functional characteristics and sites of gene expression of the α1, β1, γ2-isoform of the rat GABAAreceptor. J Neurosci 10:2330–2337.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Nayeem N,
    2. Green TP,
    3. Martin IL,
    4. Barnard EA
    (1994) Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis. J Neurochem 62:815–818.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Newland CF,
    2. Colquhoun D,
    3. 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.
    OpenUrlPubMed
  31. ↵
    1. Pedersen SE,
    2. Cohen JB
    (1990) d-Tubocurarine binding sites are located at α-γ and α-δ subunit interfaces of the nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 87:2785–2789.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Pollard S,
    2. Thompson CL,
    3. Stephenson FA
    (1995) Quantitative characterization of α6 and α1α6 subunit-containing native γ-aminobutyric acid A receptors of adult rat cerebellum demonstrates two alpha subunits per receptor oligomer. J Biol Chem 270:21285–21290.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Pritchett DB,
    2. Seeburg PH
    (1991) γ-Aminobutyric acid type A receptor point mutation increases the affinity of compounds for the benzodiazepine site. Proc Natl Acad Sci USA 88:1421–1425.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Pritchett DB,
    2. Sontheimer H,
    3. Shivers BD,
    4. Ymer S,
    5. Kettenmann H,
    6. Schofield PR,
    7. Seeburg PH
    (1989) Importance of a novel GABAAreceptor subunit for benzodiazepine pharmacology. Nature 338:582–585.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Quirk K,
    2. Gillard NP,
    3. Ragan CI,
    4. Whiting PJ,
    5. McKernan RM
    (1994) γ-aminobutyric acid type A receptors in the rat brain can contain both γ2 and γ3 subunits, but γ1 does not exist in combination with another γ subunit. Mol Pharmacol 45:1061–1070.
    OpenUrlAbstract
  36. ↵
    1. Raftery MA,
    2. Hunkapiller MW,
    3. Strader CD,
    4. Hood LE
    (1980) Acetylcholine receptor: complex of homologous subunits. Science 208:1454–1457.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Revah F,
    2. Bertrand D,
    3. Galzi J-L,
    4. Devillers-Thiery A,
    5. Mulle C,
    6. Hussy N,
    7. Bertrand S,
    8. Ballivet M,
    9. Changeux J-P
    (1991) Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353:846–849.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Reynolds JA,
    2. Karlin A
    (1978) Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica . Biochemistry 17:2035–2038.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Ruano D,
    2. Araujo F,
    3. Machado A,
    4. De Blas AL,
    5. Vitorica J
    (1994) Molecular characterization of type I GABAA receptor complex from rat cerebral cortex and hippocampus. Mol Brain Res 25:225–233.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Saiki RK,
    2. Gelfand DH,
    3. Stoffel DH,
    4. Scharf SJ,
    5. Higuchi R,
    6. Horn GT,
    7. Mullis KB,
    8. Erlich HA
    (1988) Primer directed enzyme amplification with a thermostable DNA polymerase. Science 239:487–491.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Sanger F,
    2. Nicklen S,
    3. Coulson AR
    (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Schofield PR,
    2. Darlison MG,
    3. Fujita N,
    4. Burt DR,
    5. Stephenson FA,
    6. Rodriguez H,
    7. Rhee LM,
    8. Ramachandran J,
    9. Reale V,
    10. Glencorse TA,
    11. Seeburg PH,
    12. Barnard EA
    (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328:221–227.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Shivers BD,
    2. Killisch I,
    3. Sprengel R,
    4. Sontheimer H,
    5. Kohler M,
    6. Schofield PR,
    7. Seeburg PH
    (1989) Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron 3:327–337.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Sigel E,
    2. Baur R,
    3. Trube G,
    4. Mohler H,
    5. Malherbe P
    (1990) The effect of subunit composition of rat brain GABAA receptor on channel function. Neuron 5:703–711.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Sigel E,
    2. Baur R,
    3. Kellenberger S,
    4. Malherbe P
    (1992) Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels. EMBO J 11:2017–2023.
    OpenUrlPubMed
  46. ↵
    1. 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.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Sine SM,
    2. Claudio T
    (1991) γ- and δ-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor. J Biol Chem 266:19369–19377.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Sine SM,
    2. Steinbach JH
    (1987) Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist. J Physiol (Lond) 385:325–359.
    OpenUrlCrossRefPubMed
    1. Smith G B,
    2. Olsen RW
    (1995) Functional domains of GABAA receptors. Trends Pharmacol Sci 16:162–168.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Twyman RE,
    2. Rogers CJ,
    3. Macdonald RL
    (1990) Intraburst kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J Physiol (Lond) 423:193–220.
    OpenUrlPubMed
  50. ↵
    1. Unwin N
    (1993) Nicotinic acetylcholine receptors at 9 A resolution. J Mol Biol 229:1101–1124.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Unwin N
    (1995) Acetylcholine receptor channel imaged in the open state. Nature 373:37–43.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Verdoorn TA
    (1994) Formation of heteromeric γ-aminobutyric acid type A receptors containing two different α subunits. Mol Pharmacol 45:475–480.
    OpenUrlAbstract
  53. ↵
    1. Verdoorn TA,
    2. Draguhn A,
    3. Ymer S,
    4. Seeburg PH,
    5. Sakmann
    (1990) Functional properties of recombinant rat GABAA receptors depend upon subunit composition. Neuron 4:919–928.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Weiss DS,
    2. 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.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Wieland HA,
    2. Luddens H,
    3. Seeburg PH
    (1992) A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J Biol Chem 267:1426–1429.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Wisden W,
    2. Laurie DJ,
    3. Monyer H,
    4. Seeburg PH
    (1992) The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 12:1040–1062.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Xu M,
    2. Akabas MH
    (1996) Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAAreceptor α1 subunit. J Gen Physiol 107:195–205.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Yakel J L,
    2. Lagrutta A,
    3. Adelman JP,
    4. 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.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Ymer S,
    2. Schofield PR,
    3. Draguhn A,
    4. Werner P,
    5. Kohler M,
    6. Seeburg PH
    (1989) GABAA receptor β subunit heterogeneity: functional expression of cloned cDNAs. EMBO J 8:1665–1670.
    OpenUrlPubMed
Back to top

In this issue

The Journal of Neuroscience: 16 (17)
Journal of Neuroscience
Vol. 16, Issue 17
1 Sep 1996
  • Table of Contents
  • Index by author
Email

Thank you for sharing this Journal of Neuroscience article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Stoichiometry of a Recombinant GABAA Receptor
(Your Name) has forwarded a page to you from Journal of Neuroscience
(Your Name) thought you would be interested in this article in Journal of Neuroscience.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Print
View Full Page PDF
Citation Tools
Stoichiometry of a Recombinant GABAA Receptor
Yongchang Chang, Ruoping Wang, Sonal Barot, David S. Weiss
Journal of Neuroscience 1 September 1996, 16 (17) 5415-5424; DOI: 10.1523/JNEUROSCI.16-17-05415.1996

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Respond to this article
Request Permissions
Share
Stoichiometry of a Recombinant GABAA Receptor
Yongchang Chang, Ruoping Wang, Sonal Barot, David S. Weiss
Journal of Neuroscience 1 September 1996, 16 (17) 5415-5424; DOI: 10.1523/JNEUROSCI.16-17-05415.1996
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • Footnotes
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Keywords

  • GABA
  • stoichiometry
  • receptor
  • ion channel
  • mutagenesis

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.

Related Articles

Cited By...

More in this TOC Section

  • Choice Behavior Guided by Learned, But Not Innate, Taste Aversion Recruits the Orbitofrontal Cortex
  • Maturation of Spontaneous Firing Properties after Hearing Onset in Rat Auditory Nerve Fibers: Spontaneous Rates, Refractoriness, and Interfiber Correlations
  • Insulin Treatment Prevents Neuroinflammation and Neuronal Injury with Restored Neurobehavioral Function in Models of HIV/AIDS Neurodegeneration
Show more Articles
  • Home
  • Alerts
  • Visit Society for Neuroscience on Facebook
  • Follow Society for Neuroscience on Twitter
  • Follow Society for Neuroscience on LinkedIn
  • Visit Society for Neuroscience on Youtube
  • Follow our RSS feeds

Content

  • Early Release
  • Current Issue
  • Issue Archive
  • Collections

Information

  • For Authors
  • For Advertisers
  • For the Media
  • For Subscribers

About

  • About the Journal
  • Editorial Board
  • Privacy Policy
  • Contact
(JNeurosci logo)
(SfN logo)

Copyright © 2023 by the Society for Neuroscience.
JNeurosci Online ISSN: 1529-2401

The ideas and opinions expressed in JNeurosci do not necessarily reflect those of SfN or the JNeurosci Editorial Board. Publication of an advertisement or other product mention in JNeurosci should not be construed as an endorsement of the manufacturer’s claims. SfN does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of any material contained in JNeurosci.