Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1

Inwardly rectifying potassium channels are important in cellular repolariza tion of many excitable tissues. Amino acid sequence alignment of different mammalian inward rectifier K+ channels revealed two absolutely conserved cy steine residues in the putative extracellular face, suggesting a possible d isulfide bond. Replacement of these cysteine residues in the Kir2.1 channel (i.e., C122 and C154) with either alanine or serine abolished current in X enopus laevis oocytes although Western blotting established that the channe ls were fully expressed. The digestion pattern of channels treated with Vs protease combined with Western blotting under reducing and nonreducing cond itions confirmed intrasubunit cross-linking of C122 and C154. Whole-cell an d single channel current recordings of oocytes expressing tandem tetrameric constructs with one or two of the mutant subunits suggested that insertion of one mutant subunit is sufficient to eliminate channel function. Coexpre ssion studies confirmed that the cysteine mutant channels eliminate wild-ty pe Kir2,1 currents in a dominant-negative manner. Despite these results, su lfhydryl reduction did not alter the functional properties of Kir2.1 curren ts. Molecular modeling of Kir2.1 with the two cysteines cross-linked predic ted that the extracellular loop between the first transmembrane domain and the pore helix contains a beta-hairpin structure. Distinct from the KcsA st ructure, the disulfide bond together with the beta-hairpin structure is exp ected to constrain and stabilize the P-loop and selectivity filter. Taken t ogether, these results suggest that intramolecular disulfide bond exists be tween C122 and C154 of Kir3.1 channel and this cross-link might be required for proper channel folding.