Investigation of the phenylalkylamine binding site in hKv1.3 (H399T), a mutant with a reduced C-type inactivated state

Summary
To screen for residues of hKv1.3 important for current block by the phenylalkylamine (PAA) verapamil, the inactivated-state-reduced H399T mutant was used as a background for mutagenesis studies. This approach was applied to mainly abolish the accumulation in the inactivated blocked state, from which recovery 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 times less pronounced for derivatives lacking the 4-methoxy group of the (3,4-dimethoxyphenyl)ethyl-methyl-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 A413 modulates the access of phenylalkylamines to their binding site depending on the occupancy of the phenylrings 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 A413. 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.
Published as:
Dreker T, Grissmer S.  2005. Investigation of the phenylalkylamine binding site in hKv1.3 (H399T), a mutant with a reduced C-type inactivated state.  Molecular Pharmacology in press