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Evidence for the formation of functionally distinct GABA_A receptors

Paul A. Davies, Ewen F. Kirkness* and Tim G. Hales

1. We transiently introduced the human GABA_A receptor subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing 132 receptors (WSS-1 cells) to establish whether the subunit competes with the 2 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 subunit cDNA.
2. The subunit did not change the potency of GABA: the GABA EC₅₀ was 34 ± 6 µM in control WSS-1 cells and 37 ± 6 µM in cells expressing the subunit. The introduction of the subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 ± 0.2 nA in control cells to 0.9 ± 0.2 nA in cells expressing the subunit (P < 0.05).
3. The subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from subunit-containing WSS-1 cells exhibited spontaneously opening GABA_A channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the subunit did not alter the GABA-evoked single-channel cord conductance.
4. The anaesthetic 2,6-diisopropylphenol (propofol, 3 µM) and the benzodiazepine flunitrazepam (1 µM) potentiated GABA-evoked currents recorded from control cells labelled with GFP. The subunit reduced potentiation by both agents 48-96 h after transfection.
5. The introduction of the subunit had no effect on the ability of propofol (3-30 µM) relative to GABA (1 mM) to activate GABA_A receptors in WSS-1 cells. High concentrations of propofol ( 100 µM) produced a more marked desensitization of GABA_A receptor activity in WSS-1 cells transfected with cDNA for the subunit than in control cells.
6. There was no difference in the potency of Zn²⁺ as an inhibitor of currents recorded from control cells (IC₅₀ = 165 ± 34 µM) or cells expressing the subunit (IC₅₀ = 179 ± 11 µM).
7. GABA-activated currents recorded both from control cells and cells expressing the subunit reversed in sign at the Cl^- equilibrium potential and exhibited outward rectification.
8. The introduction of the subunit changes the functional properties of GABA_A receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of , , and subunits.

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