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

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 alpha 1, beta 1, gamma 2S and delta types, and by using cultured rat cerebellar granule neurones. 2. The potency of Zn2+ as a non-competitive antagonist of GABA-activated responses on alpha 1 beta 1 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 mu M. Zinc-induced inhibition was largely unaffected by alkaline pH up to pH 9.4. 3. For alpha 1 beta 1 delta 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 mu M. Acidification of Ringer solution resulted in a reduced antagonism by Zn2+ (IC50, 49.0 mu M) without affecting the type of inhibition. At pH 9.4, Zn2+ inhibition remained unaffected. 4. The addition of the gamma 2S subunit to the alpha 1 beta 1 delta construct caused a marked reduction in the potency of Zn2+ (IC50, 615 mu M), comparable to that observed with alpha 1 beta 1 gamma 2S receptors (IC50 639 mu M). 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 mu M. 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 alpha 1 beta 1 delta receptors is shared by GABA(A) receptors on cerebellar granule neurones, which are known to express delta-subunit-containing receptors. This novel mechanism is masked when a gamma 2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABA(A) receptors to endogenous cations.