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The Role of an α Subtype M2-M3 His in Regulating Inhibition of GABAA Receptor Current by Zinc and Other Divalent Cations

Sensitivity of GABAA receptors (GABARs) to inhibition by zinc and other divalent cations is influenced by the α subunit subtype composition of the receptor. For example, α6β3γ2L receptors are more sensitive to inhibition by zinc than α1β3γ2L receptors. We examined the role of a His residue located in the M2-M3 extracellular domain (rat α6 H273) in the enhanced zinc sensitivity conferred by the α6 subtype. The α1 subtype contains an Asn (N274) residue in the equivalent location. GABA-activated whole-cell currents were obtained from L929 fibroblasts after transient transfection with expression vectors containing GABAA receptor cDNAs. Mutation of α1 (α1(N274H)) or α6 (α6(H273N)) subtypes did not alter the GABA EC50 of αβ3γ2L receptors. α1(N274H)β3γ2L receptor currents were as sensitive to zinc as α6β3γ2L receptor currents, although α6(H273N)β3γ2L receptor currents had the reduced zinc sensitivity of α1β3γ2L receptor currents. We also examined the activity of other inhibitory divalent cations with varying α subtype dependence: nickel, cadmium, and copper. α6β3γ2L receptor currents were more sensitive to nickel, equally sensitive to cadmium, and less sensitive to copper than α1β3γ2L receptor currents. Studies with α1 and α6 chimeric subunits indicated that the structural dependencies of the activity of some of these cations were different from zinc. Compared with α6β3γ2L receptor currents, α6(H273N)β3γ2L receptor currents had reduced sensitivity to cadmium and nickel, but the sensitivity to copper was unchanged. Compared with α1β3γ2L receptor currents, α1(N274H)β3γ2L receptor currents had increased sensitivity to nickel, but the sensitivity to cadmium and copper was unchanged. These findings indicate that H273 of the α6 subtype plays an important role in determining the sensitivity of recombinant GABARs to the divalent cations zinc, cadmium, and nickel, but not to copper. Our results also suggest that the extracellular N-terminal domain of the α1 subunit contributes to a regulatory site(s) for divalent cations, conferring high sensitivity to inhibition by copper and cadmium.