Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABA(A) 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.