Hc. Cho et al., Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1, BIOCHEM, 39(16), 2000, pp. 4649-4657
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.