The Role of an α Subtype M2-M3His in Regulating Inhibition of GABAAReceptor Current by Zinc and Other Divalent Cations

Abstract

Sensitivity of GABA_Areceptors (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-M₃extracellular 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 GABA_Areceptor cDNAs. Mutation of α1 (α1_(N274H)) or α6 (α6_(H273N)) subtypes did not alter the GABA EC₅₀of 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.