Molecular mapping of a site for Cd²⁺‐induced modification of human ether‐à‐go‐go‐related gene (hERG) channel activation

Cd(2+) slows the rate of activation, accelerates the rate of deactivation and shifts the half-points of voltage-dependent activation (V(0.5,act)) and inactivation (V(0.5,inact)) of human ether-à-go-go-related gene (hERG) K(+) channels. To identify specific Cd(2+)-binding sites on the hERG channel, we mutated potential Cd(2+)-coordination residues located in the transmembrane domains or extracellular loops linking these domains, including five Cys, three His, nine Asp and eight Glu residues. Each residue was individually substituted with Ala and the resulting mutant channels heterologously expressed in Xenopus oocytes and their biophysical properties determined with standard two-microelectrode voltage-clamp technique. Cd(2+) at 0.5 mM caused a +36 mV shift of V(0.5,act) and a +18 mV shift of V(0.5,inact) in wild-type channels. Most mutant channels had a similar sensitivity to 0.5 mM Cd(2+). Mutation of single Asp residues located in the S2 (D456, D460) or S3 (D509) domains reduced the Cd(2+)-induced shift in V(0.5,act), but not V(0.5,inact). Combined mutations of two or three of these key Asp residues nearly eliminated the shift induced by 0.5 mM Cd(2+). Mutation of D456, D460 and D509 also reduced the comparatively low-affinity effects of Ca(2+) and Mg(2+) on V(0.5,act). Extracellular Cd(2+) modulates hERG channel activation by binding to a coordination site formed, at least in part, by three Asp residues.