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An N-Terminal Histidine Is the Primary Determinant of Subunit-Dependent Cu²⁺ Sensitivity of 32L GABA_A Receptors

Department of Neurology, University of Michigan Health System, University of Michigan, Ann Arbor, Michigan (H.K.); and Departments of Neurology, Molecular Physiology and Biophysics, and Pharmacology, Vanderbilt University, Nashville, Tennessee (R.L.M.)

Copper (Cu²⁺) is a physiologically important cation and is released from nerve terminals. Cu²⁺ modulates GABA_A receptor currents in an subunit subtype-dependent manner; 132L receptors are more sensitive to Cu²⁺ than 632L receptors. We compared the effect of Cu²⁺ on 32L receptors containing each of the six subtypes and generated 1/6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for subtype-dependent differences in Cu²⁺ sensitivity. Whole-cell GABA_A receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant subunits with 3 and 2L subunits. Maximal Cu²⁺ inhibition of 132L and 232L receptor currents was larger (52.2 ± 3.0 and 59.0 ± 2.5%, respectively) than maximal inhibition of 332L, 432L, 532L, and 632L 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 1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu²⁺ to an extent similar to those with 1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu²⁺ sensitivity. 1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the 2 subtype) conferred higher Cu²⁺ sensitivity, and the H141 residue was the major determinant in the motif. The 3 subtype M2 domain residue H267, which is a major determinant of Zn²⁺ inhibition, and 6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn²⁺ sensitivity of the 6 subtype, also seemed to contribute to Cu²⁺ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in 1 and 2 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.

Address correspondence to: Dr. Robert L. Macdonald, Department of Neurology, Vanderbilt University, 2100 Pierce Ave., Nashville, TN 37212 (E-mail: robert.macdonald{at}vanderbilt.edu).