Differential sensitivity of Kir2 inward-rectifier potassium channels to a mitochondrial uncoupler: identification of a regulatory site.

The aim of this study was to gain insight into the mechanism by which members of the K(ir)2 subfamily are differentially sensitive to agents that inhibit mitochondrial function by identifying responsible site(s) in K(ir)2 proteins. K(ir)2 channels were expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp and patch clamp. Incubation of oocytes in carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, inhibited K(ir)2.2 and K(ir)2.3, but not K(ir)2.1. Replacement of the first 44 amino acids of K(ir)2.2 the or of first 19 K(ir)2.3 with the first 45 of K(ir)2.1 did not affect the sensitivity of the channels to FCCP. In contrast, a larger substitution of K(ir)2.1 N-terminal sequence (1-78) into K(ir)2.2 or K(ir)2.3 produced channels that were resistant to FCCP. Sequence alignment between residues 46 and 78 (K(ir)2.1 numbering) revealed four residues that are the same in K(ir)2.2 and K(ir)2.3 but different in K(ir)2.1. Each of these four residues in the resistant chimera was converted back to the K(ir)2.2/K(ir)2.3 amino acid. Three of the mutants (D51N, I59A, and G65S) were not sensitive to FCCP, but the H53Q mutant was sensitive. K(ir)2.1-H53A and K(ir)2.1-H53E were also sensitive. In contrast, K(ir)2.1-H53R and K(ir)2.1-H53K were recovered during resistant. K(ir)2.2 and K(ir)2.3 currents perfusion of inside-out patches from FCCP-treated oocytes. FCCP was without effect on K(ir)2.2 and K(ir)2.3 when applied directly to inside-out patches. Together, these results suggest inhibition of K(ir)2.2 and K(ir)2.3 by a ligand that bears a positive charge and is produced by an intracellular action of FCCP.