Abstract

To screen for residues of hKv1.3 important for current block by the phenylalkylamine verapamil, the inactivated-state-reduced H399T mutant was used as a background for mutagenesis studies. This approach was applied mainly to abolish the accumulation in the inactivated blocked state, recovery from which in the wild type is normally slow. Substitution of amino acids in the S6 transmembrane helix indicated a heavy disruption of verapamil block by the A413C mutation, reducing the IC₅₀ from 2.4 to 267 μM. Subsequent scanning for verapamil moieties essential for current block was performed by application of derivatives with altered side groups. Neither the removal of the nitrile or the methyl group nor the addition of a methoxy group resulted in major variations of IC₅₀ values for hKv1.3 (H399T) current block. However, disruption of current block by A413C was 4- to 10-fold less pronounced for derivatives lacking the 4-methoxy group of the (3,4-dimethoxyphenyl)ethylmethyl-amino part (devapamil) or all four methoxy groups (emopamil), respectively. Emopamil displayed a Hill coefficient of 2 for hKv1.3 (H399T/A413C) instead of 1 for hKv1.3 (H399T) current block. These results might indicate that the alteration of Ala413 modulates the access of phenylalkylamines to their binding site depending on the occupancy of the phenyl rings with methoxy groups. A computer-based docking model shows a subset of docked PAA conformations, with a spatial proximity between the (4-methoxyphenyl)ethyl-methyl-amino group and Ala413. The PAA binding site might therefore include a binding pocket for the aromatic ring of the ethyl-methyl-amino part in an S6–S6 interface gap.