Abstract
The aim of this study was to gain insight into the mechanism by which members of the Kir2 subfamily are differentially sensitive to agents that inhibit mitochondrial function by identifying responsible site(s) in Kir2 proteins. Kir2 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 Kir2.2 and Kir2.3, but not Kir2.1. Replacement of the first 44 amino acids of Kir2.2 the or of first 19 Kir2.3 with the first 45 of Kir2.1 did not affect the sensitivity of the channels to FCCP. In contrast, a larger substitution of Kir2.1 N-terminal sequence (1-78) into Kir2.2 or Kir2.3 produced channels that were resistant to FCCP. Sequence alignment between residues 46 and 78 (Kir2.1 numbering) revealed four residues that are the same in Kir2.2 and Kir2.3 but different in Kir2.1. Each of these four residues in the resistant chimera was converted back to the Kir2.2/Kir2.3 amino acid. Three of the mutants (D51N, I59A, and G65S) were not sensitive to FCCP, but the H53Q mutant was sensitive. Kir2.1-H53A and Kir2.1-H53E were also sensitive. In contrast, Kir2.1-H53R and Kir2.1-H53K were recovered during resistant. Kir2.2 and Kir2.3 currents perfusion of inside-out patches from FCCP-treated oocytes. FCCP was without effect on Kir2.2 and Kir2.3 when applied directly to inside-out patches. Together, these results suggest inhibition of Kir2.2 and Kir2.3 by a ligand that bears a positive charge and is produced by an intracellular action of FCCP.
- Received October 14, 2004.
- Accepted January 3, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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