Expression of functional receptors by the human gamma-aminobutyric acid A gamma 2 subunit

Abstract

gamma-Aminobutyric acid A (GABA(A)) receptors are heteromeric membrane proteins formed mainly by various combinations of alpha, beta, and gamma subunits; and it is commonly thought that the gamma 2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the gamma 2L 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 gamma 2L receptors were also activated by beta-alanine > taurine > glycine, and, like some types of heteromeric GABA(A) receptors, the gamma 2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human gamma 2L 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.

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.

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.

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.

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.

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.