An N-terminal histidine is the primary determinant of alpha subunit-dependent Cu2+ sensitivity of alphabeta3gamma2L GABA(A) receptors.
Copper (Cu2+) is a physiologically important cation and is released from nerve terminals. Cu2+ modulates GABAA receptor currents in an alpha subunit subtype-dependent manner; alpha1beta3gamma2L receptors are more sensitive to Cu2+ than alpha6beta3gamma2L receptors. We compared the effect of Cu2+ on alphabeta3gamma2L receptors containing each of the six alpha subtypes and generated alpha1/alpha6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for alpha subtype-dependent differences in Cu2+ sensitivity. Whole-cell GABAA receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant alpha subunits with beta3 and gamma2L subunits. Maximal Cu2+ inhibition of alpha1beta3gamma2L and alpha2beta3gamma2L receptor currents was larger (52.2 +/- 3.0 and 59.0 +/- 2.5%, respectively) than maximal inhibition of alpha3beta3gamma2L, alpha4beta3gamma2L, alpha5beta3gamma2L, and alpha6beta3gamma2L 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 alpha1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu2+ to an extent similar to those with alpha1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu2+ sensitivity. alpha1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the alpha2 subtype) conferred higher Cu2+ sensitivity, and the H141 residue was the major determinant in the motif. The beta3 subtype M2 domain residue H267, which is a major determinant of Zn2+ inhibition, and alpha6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn2+ sensitivity of the alpha6 subtype, also seemed to contribute to Cu2+ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in alpha1 and alpha2 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 GABAA receptors at sites similar to or overlapping Zn2+ sites.
Department of Neurology, University of Michigan, Ann Arbor, MI, USA.
Heejeong
H
Kim
Robert L
RL
Macdonald
D000595:Amino Acid Sequence; D000818:Animals; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D003300:Copper / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid
D002412:Cations; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D006639:Histidine; D003300:Copper