Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain

The mechanism of verapamil block of the delayed rectifier K currents (IK(DR)) in chick dorsal root ganglion (DRG) neurons was investigated using the whole-cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890. Block by D890 displayed similar characteristics to that of verapamil, indicating the same state-dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e-fold change of the on rate). Given that verapamil block is insensitive to voltage (Trequattrini et al., 1998), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm. In external K and saturating verapamil we recorded tail currents that did not decay monotonically but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested (DeCoursey, 1995) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890. The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium, and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies. British Journal of Pharmacology (1999) 126, 1699–1706; doi:10.1038/sj.bjp.0702477