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Evidence for the formation of functionally distinct αβγε GABA A receptors
Author(s) -
Davies Paul A.,
Kirkness Ewen F.,
Hales Tim G.
Publication year - 2001
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2001.0101k.x
Subject(s) - protein subunit , gabaa receptor , transfection , patch clamp , hek 293 cells , microbiology and biotechnology , receptor , biology , chemistry , biophysics , cell culture , biochemistry , gene , genetics
1 We transiently introduced the human GABA A receptor ε subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing α1β3γ2 receptors (WSS‐1 cells) to establish whether the subunit competes with the γ2 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 ε subunit cDNA. 2 The ε subunit did not change the potency of GABA: the GABA EC 50 was 34 ± 6 μ m in control WSS‐1 cells and 37 ± 6 μ m in cells expressing the ε subunit. The introduction of the ε subunit reduced the peak current amplitude activated by GABA (1 m m ) from 1.8 ± 0.2 nA in control cells to 0.9 ± 0.2 nA in cells expressing the ε subunit ( P < 0.05 ). 3 The ε subunit caused the appearance of leak currents recorded in the absence of GABA. Outside‐out patches excised from ε 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 ε subunit did not alter the GABA‐evoked single‐channel cord conductance. 4 The anaesthetic 2,6‐diisopropylphenol (propofol, 3 μ m ) and the benzodiazepine flunitrazepam (1 μ m ) potentiated GABA‐evoked currents recorded from control cells labelled with GFP. The ε subunit reduced potentiation by both agents 48–96 h after transfection. 5 The introduction of the ε subunit had no effect on the ability of propofol (3‐30 μ m ) relative to GABA (1 m m ) to activate GABA A receptors in WSS‐1 cells. High concentrations of propofol ( > 100 μ m ) produced a more marked desensitization of GABA A receptor activity in WSS‐1 cells transfected with cDNA for the ε subunit than in control cells. 6 There was no difference in the potency of Zn 2+ as an inhibitor of currents recorded from control cells ( IC 50 = 165 ± 34 μ m ) or cells expressing the ε subunit (IC 50 = 179 ± 11 μ m ). 7 GABA‐activated currents recorded both from control cells and cells expressing the ε subunit reversed in sign at the Cl − equilibrium potential and exhibited outward rectification. 8 The introduction of the ε 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 α, β, γ and ε subunits.