Mol Pharmacol 2000 Feb 01;572:367-74.

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

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This study reports that the affinity of HERG1 A for dofetilide is decreased from 0.125 +/- 0.003 microM for wild-type (WT) channels to 15 +/- 3 microM for F656V, a mutation in the COOH-terminal half of the S6. Similarly, the IC(50) for quinidine was increased from 8 +/- 4 microM for WT to 219 +/- 65 microM for the F656V mutation, whereas affinity for external tetraethylammonium was similar for WT (51 +/- 10 mM) and F656V (36 +/- 10 mM, NS). Kinetics of onset of inactivation of F656V was similar to WT but kinetics of deactivation, activation, and recovery from inactivation differed from WT. However, mutations in nearby amino acids in the S6 more strikingly altered deactivation, activation, and recovery from inactivation but had little effect on affinity for dofetilide. To assess the effects of disruption of inactivation, the S631A mutation was made. The S631A mutation altered the IC(50) for dofetilide to 20 +/- 3 microM, but the IC(50) for quinidine was unchanged at 8 +/- 4 microM for WT and 10 +/- 1 microM for S631A. To address whether the F656V mutation alters the IC(50) for dofetilide in a channel that does not inactivate, the double mutation S631A/F656V was made. The IC(50) for dofetilide of the double mutation was 32 +/- 3 microM, which is not substantially different than that of S631A. These data support the notion that allosteric changes occurring during the process of inactivation are necessary for high-affinity dofetilide binding. In conclusion, the Phe-656 residue of HERG is a molecular determinant of high-affinity dofetilide binding.

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Species referenced: Xenopus laevis
Genes referenced: kcnh2