An N-terminal histidine regulates Zn2+ inhibition on the murine GABAA receptor β3 subunit

1 Whole-cell currents were recorded from Xenopus laevis oocytes and human embryonic kidney cells expressing GABA(A) receptor beta3 subunit homomers to search for additional residues affecting Zn2 + inhibition. These residues would complement the previously identified histidine (H267), present just within the external portal of the ion channel, which modulates Zn2 + inhibition. 2 Zinc inhibited the pentobarbitone-gated current on beta3(H267A) homomers at pH 7.4, but this effect was abolished at pH 5.4. The Zn2 + -sensitive spontaneous beta3 subunit-mediated conductance was also insensitive to block by Zn2 + at pH 5.4. 3 Changing external pH enabled the titration of the Zn2 + 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 beta3 receptor subunit were substituted with alanine, in addition to the background mutation, H267A, to assess their sensitivity to Zn2+ inhibition. The Zn2+ IC50 was unaffected by either the H119A or H191A mutations. 5 The remaining histidine, H107, the only other candidate likely to participate in Zn2+ inhibition, was substituted with various residues. Most mutants were expressed at the cell surface but they disrupted functional expression of beta3 homomers. However, H107G was functional and demonstrated a marked reduction in sensitivity to Zn2 +. 6 GABAA receptor beta3 subunits form functional ion channels that can be inhibited by Zn2+. Two histidine residues are largely responsible for this effect, H267 in the pore lining region and H107 residing in the extracellular N-terminal domain.