Molecular determinant of high-affinity dofetilide binding to HERG1 expressed in Xenopus oocytes: Involvement of S6 sites

aThis study reports that the affinity of HERG1 A for dofetilide is decrease d from 0.125 +/- 0.003 mu M for wild-type (WT) channels to 15 +/- 3 mu M fo r F656V, a mutation in the COOH-terminal half of the S6. Similarly, the IC5 0 for quinidine was increased from 8 +/- 4 mu M for WT to 219 +/- 65 mu M f or the F656V mutation, whereas affinity for external tetraethylammonium was similar for WT (51 +/- 10 mM) and F656V (36 +/- 10 mM, NS). Kinetics of on set of inactivation of F656V was similar to WT but kinetics of deactivation , activation, and recovery from inactivation differed from WT. However, mut ations in nearby amino acids in the S6 more strikingly altered deactivation , activation, and recovery from inactivation but had little effect on affin ity for dofetilide. To assess the effects of disruption of inactivation, th e S631A mutation was made. The S631A mutation altered the IC50 for dofetili de to 20 +/- 3 mM, but the IC50 for quinidine was unchanged at 8 +/- 4 mu M for WT and 10 +/- 1 mu M for S631A. To address whether the F656V mutation alters the IC50 for dofetilide in a channel that does not inactivate, the d ouble mutation S631A/F656V was made. The IC50 for dofetilide of the double mutation was 32 +/- 3 mu M, which is not substantially different than that of S631A. These data support the notion that allosteric changes occurring d uring the process of inactivation are necessary for high-affinity dofetilid e binding. In conclusion, the Phe-656 residue of HERG is a molecular determ inant of high-affinity dofetilide binding.