1. The γ-subunit in recombinant γ-aminobutyric acid (GABA(A)) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2+ effects on macroscopic and single-channel currents of recombinant α1β2 and α1β2γ2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 μM)-triggered currents of α1β2 receptors in a concentration-dependent manner (inhibition equation parameters:concentration at half-amplitude (IC50) = 0.94 μM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The γ2 subunit (α1β2γ2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 μM, S = 0.86); increased GABA concentration (100 μM) antagonized inhibition by reducing apparent affinity (IC50 = 322 μM, S= 0.79). Zn2+ slowed macroscopic gating of α1β2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For α1β2γ2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of α1β2 receptors, but had little effect on currents from α1β2γ2 receptors. 4. Steady-state fluctuation analysis of macroscopic α1β2γ2 currents (n = 5) resulted in control (2 μM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 ± 5.6 and 1.41 ± 0.15 ms, means ± S.E.M.). Zn2+ (200 μM) reduced the total power almost sixfold and accelerated the slow (23 ± 2.8 ms, P < 0.05) without altering the fast (1.40 ± 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 ± 0.55; Zn2+, 4.9 ± 0.37, P < 0.05). 5. Zn2+ (500 μM) depression of previously activated current amplitudes (% control) for α1β2γ2 receptors was independent of GABA concentration (5 μM, 13.2 ± 0.72%; 100 μM, 12.2 ± 2.9%, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated α1β2 receptors with current depression more than fourfold less sensitive (5 μM GABA, IC50 = 3.8 μM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABA(A), receptors in which Zn2+ hinds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the γ-subunit.
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