Abstract

Copper (Cu²⁺) is a physiologically important cation and is released from nerve terminals. Cu²⁺ modulates GABA_A receptor currents in an α subunit subtype-dependent manner; α1β3γ2L receptors are more sensitive to Cu²⁺ than α6β3γ2L receptors. We compared the effect of Cu²⁺ on αβ3γ2L receptors containing each of the six α subtypes and generated α1/α6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for α subtype-dependent differences in Cu²⁺ sensitivity. Whole-cell GABA_A receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant α subunits with β3 and γ2L subunits. Maximal Cu²⁺ inhibition of α1β3γ2L and α2β3γ2L receptor currents was larger (52.2 ± 3.0 and 59.0 ± 2.5%, respectively) than maximal inhibition of α3β3γ2L, α4β3γ2L, α5β3γ2L, and α6β3γ2L receptor currents (22.6 ± 3.1, 19.2 ± 3.4, 20.2 ± 4.8, and 21.2 ± 3.6%, respectively). Receptors containing chimeric constructs with α1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu²⁺ to an extent similar to those with α1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu²⁺ sensitivity. α1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the α2 subtype) conferred higher Cu²⁺ sensitivity, and the H141 residue was the major determinant in the motif. The β3 subtype M2 domain residue H267, which is a major determinant of Zn²⁺ inhibition, and α6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn²⁺ sensitivity of the α6 subtype, also seemed to contribute to Cu²⁺ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in α1 and α2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu²⁺, that residue H141 is the major determinant in that motif, and that Cu²⁺ may also interact with GABA_A receptors at sites similar to or overlapping Zn²⁺ sites.