Block of the lymphocyte K⁺ channel mKv1.3 by the phenylalkylamine verapamil: Kinetic aspects of block and disruption of accumulation of block by a single point mutation

Abstract

1 Phenylalkylamines (PAA) usually known for their action on L-type Ca 2+ channels potently block the C-type inactivating lymphocyte Kv1.3 channel resulting in inhibition of activation of T lymphocytes. In order to design PAAs blocking Kv1.3 speci®cally over L-type Ca 2+ channels, we investigated the state-dependent manner of mKv1.3 block by the PAA verapamil. 2 Verapamil seems to have access to the open state (OB) and, once bound to the channel, the channel-verapamil complex is absorbed into a slowly recovering state. This state was proposed to be the inactivated blocked state (IB). Here we present a quantitative description of the transition into this state and provide evidence for the IB state through experiments with an inactivation lacking mutant channel. Since the inactivated state cannot be reached in this case the IB state cannot be reached either. 3 We show that the transition OB?IB is accelerated by verapamil most likely through a mechanism involving the reduction of [K + ] at an inactivation modulating low anity binding site for K + at the outer vestibule. 4 Measurements of the voltage-dependence of the o-rate constants for verapamil suggest that verapamil can reach the channel in its neutral form and might get partially protonated while bound. Thus only those verapamil molecules that are protonated can more easily dissociate at hyperpolarizing voltages. 5 Since open block kinetics were shown to be similar for wild type mKv1.3 and the H404T mutant mKv1.3 channel, and since the block of the H404T mutant channels by verapamil could be described exactly by a simple three-state open block model, the mutant channel could serve as a screening channel to determine open block anities of new PAA derivatives in high through-put experiments.

State-Dependent Verapamil Block of the Cloned Human Cav3.1 T-Type Ca2+ Channel

“…The observation that verapamil block was independent of the current-eliciting potential was unexpected but is consistent with results obtained for verapamil block of K ϩ channels (DeCoursey, 1995;Robe and Grissmer, 2000). One possibility is that verapamil binds outside of the transmembrane electric field.…”

“…However, the lack of a substantial drug effect on the time course of T-channel currents suggests that the kinetics of inhibition are distinct from those observed for other channel targets. It is noteworthy that verapamil did not accelerate current decay as has been reported in L channels (Johnson et al, 1996;Dilmac et al, 2004) and K ϩ channels (DeCoursey, 1995;Catacuzzeno et al, 1999;Robe and Grissmer, 2000); in addition, the drug unbinding reaction could not be observed in the tail currents (data not shown) as in K ϩ channels (DeCoursey, 1995;Catacuzzeno et al, 1999;Robe and Grissmer, 2000). The dependence on holding potential was strongly suggestive of high-affinity drug binding to an inactivated state of the channel.…”

“…This finding suggested that the primary active species of drug was probably the uncharged form, which may bind within the field (in the pore) with no observable voltage-dependence. A similar mechanism has been proposed for verapamil inhibition of K ϩ channels (DeCoursey, 1995;Catacuzzeno et al, 1999;Robe and Grissmer, 2000).…”

“…It has been reported that verapamil accesses its binding site from the intracellular space and binds in the pore of both L-type Ca 2ϩ channels and K ϩ channels (DeCoursey, 1995;Johnson et al, 1996;Robe and Grissmer, 2000;Dilmac et al, 2004). We hypothesized that verapamil would also bind in the pore of the T channel and predicted that it would compete with the permeant ion for a binding site.…”

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

“…Indeed, clinical trials of Ca 2ϩ -active reagents have already been reported (50, 56 -58), e.g., verapamil, a HVA Ca 2ϩ channel blocker, has been used in vitro and in combined therapy approaches (48 -52). At higher doses, verapamil also blocks potassium channels (59), which may lead to some ambiguity in its mechanism of action. CAI is a promising anticancer agent acting on NVG channels, which is in Phase I and II clinical trials (56 -58).…”