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

Cd2+ slows the rate of activation, accelerates the rate of deactivation and shifts the half-points of voltage-dependent activation (V-0.5,V-act) and inactivation (V-0.5,V-inact) of human ether-a-go-go-related gene (hERG) K+ channels. To identify specific Cd2+-binding sites on the hERG channel, we mutated potential Cd2+-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. Cd2+ at 0.5 mM caused a +36 mV shift of V-0.5,V-act and a +18 mV shift of V-0.5,V-inact in wild-type channels. Most mutant channels had a similar sensitivity to 0.5 mM Cd2+. Mutation of single Asp residues located in the S2 (D456, D460) or S3 (D509) domains reduced the Cd2+-induced shift in V-0.5,V-act,V- but not V-0.5,V-inact. Combined mutations of two or three of these key Asp residues nearly eliminated the shift induced by 0.5 mM Cd2+. Mutation of D456, D460 and D509 also reduced the comparatively low-affinity effects of Ca2+ and Mg2+ on V-0.5,V-act. Extracellular Cd2+ modulates hERG channel activation by binding to a coordination site formed, at least in part, by three Asp residues.