Evidence for the formation of functionally distinct alpha beta gamma epsilon GABA(A) receptors

1. We transiently introduced the human GABA, receptor e subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing alpha1 beta3 gamm a2 receptors (WSS-1 cells) to establish, whether the subunit competes with the gamma2 subunit for assembly into receptors.,. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the c subunit cDNA. 2. The c subunit, did not change the potency of GABA: the GABA EC50 was 34 +/- 6 mum in control WSS-1 cells and 37 +/- 6 mum in cells expressing the e psilon subunit. The introduction of the epsilon subunit reduced the peak cu rrent amplitude activated by GABA (1 mM) from 1.8 +/- 0.2 nA in control cel ls to 0.9 +/- 0.2 nA in cells expressing the epsilon subunit (P < 0.05). 3. The epsilon subunit, caused the appearance of leak currents recorded in the absence of GABA. Out,side-out patches excised from epsilon subunit-cont aining WSS-1 cells exhibited spontaneously, opening GABA(A) channels not se en in patches excised from control GFP-expressing WSS-1 cells. Introduction of the epsilon subunit did not alter the GABA-evoked single-channel cord c onductance. 4. The anaesthetic 2,6-diisopropylphenol (propofol, 3,um) and the benzodiaz epine flunitrazepam (1 pm) potentiated GABA-evoked currents recorded from c ontrol cells labelled with GFP. The c subunit reduced potentiation by both agents 48-96 h after transfection. 5. The introduction of the epsilon subunit had no effect on the ability of propofol (3-30 mum) relative to GABA (1 mM) to activate GABAA receptors in WSS-1 cells. High concentrations of propofol (greater than or equal to 100 mum) produced a inore marked desensitization of GABA(A) receptor activity i n WSS-1 cells transfected k ith cDNA for the r subunit than in control cell s. 6. There was no difference in the potency of Zn2+ as an inhibitor of curren ts recorded from control cells (IC50 = 165 +/- 34 muM) or cells expressing the epsilon subunit (IC50 = 179 +/- 11 muM). 7. GABA-activated currents recorded both froin control cells and cells expr essing the e subunit reversed in sign at the Cl- equilibrium potential and exhibited outward rectification. 8. The introduction of the epsilon subunit chanLyes the functional properti es of GABA(A) receptors in WSS-1 cells. The resulting receptors have a uniq ue combination of properties indicative of the co-assembly of alpha, beta, gamma and epsilon subunits.