State-Dependent Verapamil Block of the Cloned Human Ca_v3.1 T-Type Ca²⁺ Channel

Abstract

Verapamil is a potent phenylalkylamine antihypertensive believed to exert its therapeutic effect primarily by blocking highvoltage-activated L-type calcium channels. It was the first clinically used calcium channel blocker and remains in clinical use, although it has been eclipsed by other calcium channel blockers because of its short half-life and interactions with other channels. In addition to blocking L-type channels, it has been reported to block T-type (low-voltage activated) calcium channels. This type of cross-reactivity is likely to be beneficial in the effective control of blood pressure. Although the interactions of T channels with a number of drugs have been described, the mechanisms by which these agents modulate channel activity are largely unknown. Most calcium channel blockers exhibit state-dependence (i.e., preferential binding to certain channel conformations), but little is known about state-dependent verapamil block of T channels. We stably expressed human Ca v 3.1 T-type channels in human embryonic kidney 293 cells and studied the state-dependence of the drug with macroscopic and gating currents. Verapamil blocked currents at micromolar concentrations at polarized potentials similar to those reported for L-type channels, although unlike for L-type currents, it did not affect current time course. The drug exhibited use-dependence and significantly slowed the apparent recovery from inactivation. Current inhibition was dependent on potential. This dependence was restricted to negative potentials, although all data were consistent with verapamil binding in the pore. Gating currents were unaffected by verapamil. We propose that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel.

Verapamil Block of T-Type Calcium Channels

“…Verapamil produces tonic block (i.e., block at the resting potential), measured with infrequent depolarizations, in both T-type and L-type channels. In heterologous expression systems, the affinity of verapamil is similar, with an ED 50 for L-type channels of ϳ10 M, versus an ED 50 of ϳ20 M for T-type channels (Johnson et al, 1996;Freeze et al, 2006). D888 is a much higher-affinity ligand for L-type channels than verapamil, with an ED 50 for closed channel block of ϳ50 nM (Johnson et al, 1996).…”

“…In particular, D888 has 5-fold higher affinity for depolarized than for resting L-type channels (Johnson et al, 1996). In T-type channels, block by verapamil is modestly use-dependent (Freeze et al, 2006). We tested the use-dependence of D888 using a train of 25-ms depolarizing pulses to Ϫ10 mV with an interpulse interval of 100 ms.…”

“…In L-type channels, the affinity of verapamil for depolarized channels is on the order of 10-fold higher than for closed channels (Johnson et al, 1996). In T-type channels, the state-dependence is less dramatic, with less than 2-fold more block of depolarized channels than closed channels in the presence of 10 M verapamil (Freeze et al, 2006). D888, also known as devapamil or desmethoxyverapamil, is a related PAA lacking one methoxy group.…”

Class IV Antiarrhythmic Agents: New Compounds Using an Old Strategy

“…In contrast, stably expressed human Ca v 3.1 T-type channels in human embryonic kidney (HEK) cells were blocked by verapamil in a use-and voltagedependent manner with an IC 50 of 21.4 µM and the drug significantly slowed the recovery from inactivation. It was suggested that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel [54]. 10 µM diltiazem (which reduced I Ca,L by 18 %) had no action on T-type calcium channels in guinea-pig ventricular myocytes [47] but in rat aorta SMCs it reduced I Ca,T with an IC 50 of 30 µM, ten times less potently than I Ca,L .…”

“…4. Prolongation of the recovery from inactivation of calcium channels was described with verapamil [54], bepridil [35], semotiadil and also with diltiazem [39]. This alteration of channel behavior can also contribute to the antiarrhythmic action of CCAs by increasing the refractory period (RP) of nodal tissues.…”