Molecular Determinants of Frequency Dependence and Ca²⁺ Potentiation of Verapamil Block in the Pore Region of Ca_v1.2

Abstract

Verapamil block of Ca v 1.2 is frequency-dependent and potentiated by Ca 2ϩ . We examined the molecular determinants of these characteristics using mutations that effect Ca 2ϩ interactions with Ca v 1.2. Mutant and wild-type Ca v 1.2 channels were transiently expressed in tsA 201 cells with ␤ 1b and ␣ 2 ␦ subunits. The four conserved glutamates that compose the Ca 2ϩ

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

“…We estimated a lower bound for verapamil affinity by measuring the inhibition of peak currents under conditions in which channels were fully available at the holding potential, fully activated at the current-eliciting potential, and the duration between consecutive pulses was long. Channels depolarized from Ϫ130 to Ϫ10 mV at a frequency of 0.2 Hz exhibited an IC 50 of 21.4 M, a value less than the affinities of 40.5 and 120 M reported for low-frequency stimulation of L-type channels in heterologous systems (Motoike et al, 1999;Dilmac et al, 2004) and similar to the values previously reported for T channels in native tissues (Kuga et al, 1990;Arnoult et al, 1998). The experimental data were fit well by a single-site binding curve, suggesting that drug binding was not a cooperative process (Fig.…”

“…1). Although verapamil has been reported to speed the decay of L-type currents (Hockerman et al, 1995(Hockerman et al, , 1997Johnson et al, 1996;Nawrath and Wegener, 1997;Motoike et al, 1999;Dilmac et al, 2004), it did not affect the T-channel current time course.…”

“…Verapamil is a clinically used phenylalkylamine antihypertensive that is believed to be somewhat selective for L-type calcium channels in native tissues, with estimated IC 50 values ranging from 600 nM in vascular smooth muscle (Kuga et al, 1990) to 3 M in ventricular myocytes (Nawrath and Wegener, 1997). However, the much greater IC 50 values of 40.5 M (Dilmac et al, 2004) and 120 M (Motoike et al, 1999) reported for L-type channels in heterologous expression systems suggest that the actual affinities in vivo may be somewhat lower. Verapamil has also been shown to block T channels, with IC 50 values of 30 M in vascular smooth muscle (Kuga et al, 1990) and 70 M in spermatogenic cells (Arnoult et al, 1998).…”

“…Verapamil has also been shown to block T channels, with IC 50 values of 30 M in vascular smooth muscle (Kuga et al, 1990) and 70 M in spermatogenic cells (Arnoult et al, 1998). T channels, like L channels, seem to be blocked in a state-dependent manner (Nawrath and Wegener, 1997;Motoike et al, 1999;Heady et al, 2001;Dilmac et al, 2004). Because T-channel blockade has been implicated in the reduction of vascular tone in vitro (Boulanger et al, 1994;Karila-Cohen et al, 1996;Lam et al, 1998;VanBavel et al, 2002;Jensen et al, 2004), the interactions of verapamil with this channel may prove to be therapeutically relevant.…”

“…Block of L-type current is accompanied by prominent use-dependence, slowing of recovery kinetics, and shift of the steady-state inactivation curve to hyperpolarized potentials (Nawrath and Wegener, 1997;Dilmac et al, 2004). Multiple pore-lining residues of the L-type channel have been implicated in phenylalkylamine binding (Hockerman et al, 1995(Hockerman et al, , 1997Johnson et al, 1996;Motoike et al, 1999;Dilmac et al, 2004), suggesting that the highaffinity binding site is located in the pore.…”