Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

Abstract

γ-Aminobutyric acid A (GABA_A) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABA_A receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl^- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABA_A receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABA_A receptors in situ require further clarification.

Fast γ-aminobutyric acid (GABA)-ergic synaptic transmission in the vertebrate nervous system is mediated by the activation of two families of GABA receptors, GABA_A and GABA_C, both of which open chloride channels and share amino acidic similarities of ≈30-33% but which differ considerably in their pharmacological and electrophysiological properties. One key difference between these two families of receptors is that the GABA_C receptors are oligohomomeric, whereas the GABA_A receptors are heteromeric, requiring the assembly of several homologous subunits (α, β, γ, etc.) (1, 2).

GABA_A receptors in the neuronal plasma membrane are believed to be pentameric structures, made up of a combination of several classes of subunits, with a potential plethora of structural diversity (2). Immunoprecipitation and immunohistochemical studies of GABA_A receptors have shown that the α1β2γ2 is the most common combination of subunits forming the GABA_A receptors in the mammalian brain (3). Moreover, the γ2 subunit is believed to be necessary for the formation of the benzodiazepine-binding site of the receptor, but not necessary for the assembly, transport, or insertion of fully functional GABA_A receptors into the neuronal plasma membrane (4-6).

Although small GABA currents are produced by the expression of GABA_Aα1 subunits alone in heterologous systems, such as Xenopus oocytes and mammalian cells in culture, expression of either the human or murine γ2 subunits alone did not produce functional GABA receptors (4, 7, 8). Moreover, studies on the heterologous homomeric expression of GABA_A subunits have yielded contradicting results, with some groups reporting successful expression of GABA gated chloride channels by either the α or β subunits and others not finding evidence of functional receptors. It is suspected that nonfunctional homomers are retained in the endoplasmic reticulum by interactions with the Ig-binding protein BiP or calnexin and are then rapidly degraded (e.g., refs. 9 and 10). By using immunocytochemistry on γ2S-tranfected mammalian cells, it has been demonstrated that when expressed alone, the γ2S subunit can access the cell surface and internalize constitutively; however, no functional receptor/channels were formed (5).

In experiments designed to assess the effects of single point mutations of the γ2L subunit on GABA receptors, we decided to reexamine first the result of injecting γ2L alone into frog oocytes. Unexpectedly, the human γ2L cDNA expressed GABA-gated channels, suggesting the formation of functional oligohomomeric γ2L receptors.

Materials and Methods

Oocyte Injection and Recordings. Xenopus oocytes were isolated from ovaries taken from anesthetized frogs (Nasco and Xenopus I). For expression experiments, we used stage V and VI oocytes that were striped of surrounding epithelial and follicular cells by using fine forceps followed by collagenase treatment (Sigma, type 1A) at 0.5 mg/ml for 30 min at room temperature in frog Ringer's solution (11). Defolliculated oocytes were then stored overnight at 16°C in Barth's solution supplied with gentamicin (0.1 mg/ml). The next day, the oocytes were injected intranuclearly with 15 nl of plasmid(s) at 1 μg/μl.

Whole membrane currents were recorded 3-5 days postinjection by using a two-microelectrode voltage-clamp technique (11). An oocyte was placed in a recording chamber (≈0.1 ml) and perfused continuously (3-7 ml/min) with frog Ringer's at room temperature. The membrane potential was usually clamped at -80 mV, and membrane currents were recorded and stored in the computer for subsequent analysis.

GABA dose/response relationships were obtained by applying GABA at 3- to 5-min intervals. The results are presented as the mean and SE. EC₅₀ and Hill number were estimated as described (12, 13). GABA current/membrane voltage relationships were obtained by applying 1-s pulses in 20-mV steps from -160 to +40 mV, to the resting oocytes and during the application of GABA. All drugs were purchased from RBI-Sigma.

Plasmid Preparation. Plasmids were manipulated by using conventional techniques (14). The GABA_A receptor subunits were donated by P. Whiting (Merk Sharp & Dohme Laboratories, Essex, England). Transcription of each subunit is driven by the strong promoter-enhancer of the human cytomegalovirus. Once we noticed the expression of functional GABA receptors after injection of the γ2L subunit alone, we proceeded to corroborate the identity of the plasmid by standard restriction analysis and by sequencing the gene from both ends of the cDNA. We also reintroduced the γ2L cDNA into Escherichia coli and isolated single colonies to avoid possible contamination with other subunits. Furthermore, before injection, we used the plasmid carrying the γ2L subunit in PCR reactions to try to detect any possible α1, β2, or β3 contaminating subunits, and consistently, only the γ2 subunit was amplified.

Results

GABA Currents Elicited by γ2L Receptors. Contrary to what we expected, the oocytes injected with the γ2L subunit cDNA alone responded to GABA by generating substantial membrane currents that resembled closely the currents generated by oocytes injected with a combination of the α1β2γ2L subunits (Fig. 1 A and B). Similar to the currents generated by heteromeric GABA receptors, made up of a variety of subunits (13, 15), the currents generated by low concentrations of GABA applied to γ2L injected oocytes were fairly well maintained; but the currents elicited by high concentrations desensitized rapidly and recovered well after washing. In fact, when GABA was applied repetitively at ≈3-min intervals, the GABA current amplitude increased progressively, sometimes more than doubling its initial amplitude (Fig. 1C).

Fig. 1.

GABA currents generated by homomeric expression of the human γ2L subunit. (A) An oocyte injected with a combination of α1β2γ2Lsubunits (2:2:1) generated a typical desensitizing-inward current when exposed to GABA. (B) An oocyte expressing γ2L alone produced currents with similar characteristics. (C) Run-up of GABA responses in an oocyte expressing γ2L. The GABA current amplitude increased during consecutive applications of GABA (1 mM, ≈20 s). Oocyte was held at -60 mV.

GABA Dose/Current Relation of γ2L Receptors. Applications of GABA to oocytes injected with a combination of the α1β2γ2L subunits generated robust inward-chloride currents, similar to those described earlier (13, 15). The GABA currents of γ2L injected oocytes were also fast-desensitizing and occasionally had amplitudes of >2 μA with 1 mM GABA. The GABA dose/current amplitude relationships were fitted well by the Hill equation with a resulting EC₅₀ of 0.31 mM (n = 4, two donors) and a Hill coefficient of 0.98 (Fig. 2).

Fig. 2.

GABA dose/current response relation for γ2 receptors. The peak currents were normalized to the maximal current (GABA 10 mM) and fitted with the Hill equation. Each point is the mean ± SEM of two to four oocytes with their membrane potential clamped at -60 mV.

GABA Current/Voltage Relation of γ2L Receptors. To identify the ion permeating through the receptor channel, the current reversal potential was determined while activating the receptor with GABA (Fig. 3). As is the case for typical heteromeric GABA_A receptors expressed in oocytes, the GABA currents produced by activation of the γ2L receptors inverted direction at about -25 mV, indicating that the currents are carried mainly by Cl^- ions (16). Furthermore, the current/voltage relation indicates a rectifying channel, similar to that formed by heteromeric GABA_A receptors (13, 15, 17) and contrasting with the nonrectifying channel gated by the GABA_C ρ1 subunit (12, 18).

Fig. 3.

Current/voltage relationship in an oocyte expressing γ2L obtained during application of 10 μM GABA. Notice the strong rectification. (Inset) Sample record from another oocyte held at -60 and with 20-mV voltage steps from -120 to +20 mV.

Some Pharmacological Characteristics of γ2L Receptors. It is well known that both GABA_A and GABA_C receptors are activated by the amino acids glycine and β-alanine (18). The same was true for the γ2L-injected oocytes. For example, in one batch of oocytes expressing γ2L receptors, small currents (≈5 nA) were elicited by 1 mM glycine, whereas 1 mM β-alanine induced larger currents (up to 105 nA; Fig. 4 A-D). The relative activation potency of these amino acids was similar to that found in the other ionotropic GABA receptors, GABA > β-ala > Gly (18), and β-alanine appears to act on the same receptors as GABA because the amplitude of the respective currents is well correlated (Fig. 4E). Moreover, taurine (1 mM), an osmoregulator amino acid known to activate some GABA_A receptor populations in the CNS (19), generated small currents (≈7 nA) in γ2L oocytes (Fig. 4). Noninjected oocytes failed to generate appreciable currents with these amino acids, except for a small current due to an oocyte glycine transporter.

Fig. 4.

Activation of homomeric γ2L receptors by amino acids. Oocyte were exposed to β-alanine (A), glycine (B), taurine (C), and GABA (D), all at 1 mM. Other amino acids (glutamate and L-aspartate) failed to induce any current. (E) Relation between β-alanine and GABA currents generated by different oocytes.

Application of bicuculline, a potent and specific GABA_A receptor antagonist, during or before the application of GABA effectively and reversibly blocked the GABA currents generated by γ2L receptors (Fig. 5). Pentobarbital facilitates GABA_A receptor ion-conductance by either increasing the channel open-time or increasing its opening frequency (20). Acting on the homomeric γ2L receptors, pentobarbital (100 μM) alone generated small currents (3-5 nA), whereas it appreciably increased the GABA currents when it was coapplied with GABA (Fig. 5B). Flunitrazepam is a benzodiazepine that is used in the short-term treatment of insomnia, as a sedative hypnotic, and as preanesthetic medication (20). The GABA currents elicited by γ2L receptors were efficiently potentiated by coapplying flunitrazepam at doses as low as 1 μM, and as for pentobarbital, the potentiation persisted for some time after washing out the flunitrazepam (Fig. 5 B and C).

Fig. 5.

Modulation of γ2L receptors. (A) The GABA_A-specific antagonist bicuculline (Bic) reduced the GABA current. (B) Pentorbarbital (PB) positively modulated the γ2L currents. Notice that the GABA current remained potentiated for some time after GABA and pentobarbital had been coapplied, and that pentobarbital applied alone generated a small current. (C) Flunitrazepam (FZ) also modulated positively the γ2L GABA currents and the potentiation remained after washing out the drug for several minutes.

Discussion

Up to now, 16 GABA_A receptors subunits have been cloned, and they exhibit differential patterns of temporal and regional expression, as well as of subcellular localization (1, 2, 21). There is currently an intense effort to define the processes that control the assembly of GABA_A receptors. Many reports have suggested that, both in vivo and in heterologous expression systems, the presence of an α and a β subunit is required to make a functional receptor/channel (5, 6), whereas the γ, δ, and other subunits are auxiliary subunits needed to form receptors with all of the functional and pharmacological properties.

Assembly of functional β2γ2 and β3γ2 receptors has been reported, whereas other studies found that coexpression of β1γ2s or β2γ2L subunits does not yield functional receptors (5, 6), and there is evidence suggesting the possible interaction of the mammalian γ2 subunit with the perch GABA_C receptors (22). Furthermore, protein-protein complex formations between the GABA_A receptor and the metabotropic dopamine D5 receptor enable mutually inhibitory functional interactions (23), mediated through the second intracellular segment of the γ2 subunit. We have not found in the literature evidence of the homomeric functional expression of the γ2 subunit from any vertebrate. Considering that relatively small amounts of other GABA_A subunits may suffice to form a functional GABA receptor, we used PCR to examine any potential contamination of our plasmids with cDNAs coding for the subunits known to interact with the γ2. We did not detect amplification of the α1, β2, or β3 subunits.

On the other hand, it is also possible that the intranuclear injection induces the expression of endogenous Xenopus GABA_A receptor subunits. We have not yet fully discarded this possibility, but we consider it somewhat remote because we have previously injected DNAs encoding for other ionotropic or G protein-coupled receptors, and the oocytes did not generate GABA currents. For example, in oocytes coinjected with the serotonin 5-HT_2A receptor and the γ2L subunit DNAs, we detected the characteristic currents activated by serotonin or by GABA, whereas in the oocytes injected only with the 5-HT_2A DNA, we did not find any GABA-generated current (data not shown).

The results presented here strongly suggest that the γ2L subunit by itself is capable of forming receptors with properties similar to those of typical heteromeric GABA_A receptors. The agonistic actions of GABA, β-alanine, glycine, and taurine mirror the profile of typical heteromeric GABA_A and of homomeric GABA_C receptors (13, 15, 18). Thus, the relative potencies of the amino acids for generating currents are well conserved in the γ2L receptor. The ionic selectivity is also preserved; this is not surprising when one considers that the region presumed to form the channel (the second transmembrane segment) would be aligned in the same manner as in an heteromeric receptor, allowing the passage of chloride ions. The marked current rectification at negative membrane potentials coincides with that found for GABA_A receptors (13, 15) and differs with that of the retinal GABA_C receptor, which rectifies little or not at all (12, 18, 24). It is interesting to note that the homomeric γ2L receptor exhibits a use-dependent gain of function (run-up) but quickly desensitizes on prolonged agonist stimulation.

The homomeric γ2L receptor was positively modulated by benzodiazepines, in agreement with the established requirement of this subunit for conveying benzodiazepine sensitivity to heteromeric GABA_A receptors (4, 20). Several lines of evidence suggest that the benzodiazepine-binding site resides in the interface between an α and a γ subunit (9). The results shown here indicate that this site may also be formed by γ2-γ2 interactions or that the benzodiazepine-binding site is not located in the presumed interface but may also be formed within a single subunit. More experiments will be needed to discriminate between these possibilities.

Although the expression of the γ2L subunit alone has not been reported in any brain area, the observations shown in this report demand a careful reexamination as well as an analysis of the role of the γ2L subunit in the heteromeric complex. For example, it is known that several pharmacological and biophysical properties of the receptor depend on the amounts of γ2L subunit expressed (7). Here, we establish that the γ2L subunit alone is sufficient to produce a GABA-gated ion channel, without the participation of other auxiliary subunits, a capacity not previously found for this subunit. Whether such a homomeric receptor actually occurs in some neurons still remains to be determined. Whatever the answer turns out be, the present experiments already suggest that further studies of homomeric receptors are required.