Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors

Abstract

1 The interaction of Zn²⁺ and H⁺ ions with GABA_A receptors was examined using Xenopus laevis oocytes expressing recombinant GABA_A receptors composed of subunits selected from α1, β1, γ2S and δ types, and by using cultured rat cerebellar granule neurones. 2 The potency of Zn²⁺ as a non-competitive antagonist of GABA-activated responses on α1β1 receptors was reduced by lowering the external pH from 7.4 to 5.4, increasing the Zn²⁺ IC₅₀ value from 1.2 to 58.3 μM. Zinc-induced inhibition was largely unaffected by alkaline pH up to pH 9.4. 3 For α1β1δ subunits, concentration-response curves for GABA were displaced laterally by Zn²⁺ in accordance with a novel mixed/competitive-type inhibition. The Zn²⁺ IC₅₀ at pH 7.4 was 16.3 μM. Acidification of Ringer solution resulted in a reduced antagonism by Zn²⁺ (IC₅₀, 49.0 μM) without affecting the type of inhibition. At pH 9.4, Zn²⁺ inhibition remained unaffected. 4 The addition of the γ2S subunit to the α1β1δ construct caused a marked reduction in the potency of Zn²⁺ (IC₅₀, 615 μM), comparable to that observed with α1β1γ2S receptors (IC₅₀ 639 μM). GABA concentration-response curves were depressed in a mixed/non-competitive fashion. 5 In cultured cerebellar granule neurones, Zn²⁺ inhibited responses to GABA in a concentration-dependent manner. Lowering external pH from 7.4 to 6.4 increased the IC₅₀ from 139 to 253 μM. 6 The type of inhibition exhibited by Zn²⁺ on cerebellar granule neurones, previously grown in high K⁺-containing culture media, was complex, with the GABA concentration-response curves shifting laterally with reduced slopes and similar maxima. The Zn²⁺-induced shift in the GABA EC₅₀ values was reduced by lowering the external pH from 7.4 to 6.4. 7 The interaction of H⁺ and Zn²⁺ ions on GABA_A receptors suggests that they share either a common regulatory pathway or coincident binding sites on the receptor protein. The apparent competitive mode of block induced by Zn²⁺ on α1β1δ receptors is shared by GABA_A receptors on cerebellar granule neurones, which are known to express δ-subunit-containing receptors. This novel mechanism is masked when a γ2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABA_A receptors to endogenous cations.