Zinc ions are concentrated in the central nervous system and regulate GABA(A) receptors, which are pivotal mediators of inhibitory synaptic neurotransmission. Zinc ions inhibit GABA(A) receptor function by an allosteric mechanism that is critically dependent on the receptor subunit composition: alphabeta subunit combinations show the highest sensitivity, and alphabetagamma isoforms are the least sensitive. Here we propose a mechanistic and structural basis for this inhibition and its dependence on the receptor subunit composition. We used molecular modeling to identify three discrete sites that mediate Zn2+ inhibition. One is located within the ion channel, and the other two are on the external amino (N)-terminal face of the receptor at the interfaces between alpha and beta subunits. We found that the characteristically low Zn2+ sensitivity of GABA(A) receptors containing the gamma2 subunit results from disruption to two of the three sites after receptor subunit co-assembly.
1 Whole-cell currents were recorded from Xenopus laevis oocytes and human embryonic kidney cells expressing GABA A receptor b3 subunit homomers to search for additional residues aecting Zn 2+ inhibition. These residues would complement the previously identi®ed histidine (H267), present just within the external portal of the ion channel, which modulates Zn 2+ inhibition. 2 Zinc inhibited the pentobarbitone-gated current on b3 H267A homomers at pH 7.4, but this eect was abolished at pH 5.4. The Zn 2+ -sensitive spontaneous b3 subunit-mediated conductance was also insensitive to block by Zn 2+ at pH 5.4. 3 Changing external pH enabled the titration of the Zn 2+ sensitive binding site or signal transduction domain. The pK a was estimated at 6.8+0.03 implying the involvement of histidine residues. 4 External histidine residues in the b3 receptor subunit were substituted with alanine, in addition to the background mutation, H267A, to assess their sensitivity to Zn 2+ inhibition. The Zn 2+ IC 50 was unaected by either the H119A or H191A mutations. 5 The remaining histidine, H107, the only other candidate likely to participate in Zn 2+ inhibition, was substituted with various residues. Most mutants were expressed at the cell surface but they disrupted functional expression of b3 homomers. However, H107G was functional and demonstrated a marked reduction in sensitivity to Zn 2+ . 6 GABA A receptor b3 subunits form functional ion channels that can be inhibited by Zn
2+. Two histidine residues are largely responsible for this eect, H267 in the pore lining region and H107 residing in the extracellular N-terminal domain.
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