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Original Article

Differential Sensitivity of K_ir2 Inward-Rectifier Potassium Channels to a Mitochondrial Uncoupler: Identification of a Regulatory Site

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon

Abstract

The aim of this study was to gain insight into the mechanism by which members of the K_ir2 subfamily are differentially sensitive to agents that inhibit mitochondrial function by identifying responsible site(s) in K_ir2 proteins. K_ir2 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_ir2.2 and K_ir2.3, but not K_ir2.1. Replacement of the first 44 amino acids of K_ir2.2 the or of first 19 K_ir2.3 with the first 45 of K_ir2.1 did not affect the sensitivity of the channels to FCCP. In contrast, a larger substitution of K_ir2.1 N-terminal sequence (1-78) into K_ir2.2 or K_ir2.3 produced channels that were resistant to FCCP. Sequence alignment between residues 46 and 78 (K_ir2.1 numbering) revealed four residues that are the same in K_ir2.2 and K_ir2.3 but different in K_ir2.1. Each of these four residues in the resistant chimera was converted back to the K_ir2.2/K_ir2.3 amino acid. Three of the mutants (D51N, I59A, and G65S) were not sensitive to FCCP, but the H53Q mutant was sensitive. K_ir2.1-H53A and K_ir2.1-H53E were also sensitive. In contrast, K_ir2.1-H53R and K_ir2.1-H53K were recovered during resistant. K_ir2.2 and K_ir2.3 currents perfusion of inside-out patches from FCCP-treated oocytes. FCCP was without effect on K_ir2.2 and K_ir2.3 when applied directly to inside-out patches. Together, these results suggest inhibition of K_ir2.2 and K_ir2.3 by a ligand that bears a positive charge and is produced by an intracellular action of FCCP.

Address correspondence to: Dr. Anthony Collins, Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331-3507. E-mail: tony.collins{at}oregonstate.edu