Z Kuras1, S Grissmer. 1. Institute of Applied Physiology, Ulm University, Ulm, Germany.
Abstract
BACKGROUND AND PURPOSE: Verapamil blocks current through the voltage-gated K(+) channel K(v)1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity. EXPERIMENTAL APPROACH: We investigated the influence of intra- and extracellular K(+) and Rb(+) on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hK(v)1.3 channels. KEY RESULTS: Verapamil affinity was highest in high intracellular K(+) concentrations ([K(+)](i)) and lowest in low [Rb(+)](i), indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K(+) and Rb(+) could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate. CONCLUSIONS AND IMPLICATIONS: Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hK(v)1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.
BACKGROUND AND PURPOSE:Verapamil blocks current through the voltage-gated K(+) channel K(v)1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity. EXPERIMENTAL APPROACH: We investigated the influence of intra- and extracellular K(+) and Rb(+) on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hK(v)1.3 channels. KEY RESULTS:Verapamil affinity was highest in high intracellular K(+) concentrations ([K(+)](i)) and lowest in low [Rb(+)](i), indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K(+) and Rb(+) could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate. CONCLUSIONS AND IMPLICATIONS: Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hK(v)1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.