Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels.

BACKGROUND/AIMS: The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated.

METHODS: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hKv1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate kon of verapamil.

RESULTS: The on-rate of verapamil to block current through hKv1.3_T419C mutant channels is similar to that obtained for hKv1.3_wt channels whereas the on-rate of verapamil to block currents through hKv1.3_L417C and hKv1.3_L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hKv1.3_L346C and the double mutant hKv1.3_L346C_L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hKv1.3_I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold.

CONCLUSIONS: We conclude that position 420 in hKv1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hKv1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hKv1.3_L346C and hKv1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.