Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs

Abstract

Antihypertensive drugs of the "calcium channel blocker" or "calcium antagonist" class have been used to establish the physiological role of L-type Ca(2+) channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacology T-type Ca(2+) channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have therapeutic advantages over selective L-type blockers. The goal of this study was to identify compounds that block the Ca(v)3.2 T-type channel with high affinity, focusing on two classes of compounds: phenylalkylamines (e.g., mibefradil) and dihydropyridines (e.g., efonidipine). Compounds were tested using a validated Ca(2+) influx assay into a cell line expressing recombinant Ca(v)3.2 channels. This study identified four clinically approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC(50) < 3 microM). In contrast, other widely prescribed dihydropyridines, such as amlodipine and nifedipine, were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concentrations, contributing to their mechanism of action.

Fig. 1.

Characteristics of the dye-based assay of Ca_v3.2 activity. A, mibefradil block of raw fluorescent signal (r.f.u., relative fluorescence units). A human embryonic kidney 293 cell was loaded with Fluo-4-AM, washed, then incubated with varying micromolar concentrations of mibefradil for 30 min at 37°C. The 96-well plate was loaded into a FLEX-Station, and fluorescence was measured for 15 s before an addition of a bolus of CaCl₂ that raised extracellular Ca²⁺ to 10 mM. Results shown are the mean ± S.E.M. of three replicates. B, structure of mibefradil. C, mibefradil dose-response measurements from 15 separate experiments.

Fig. 2.

Block by verapamil, its analogs, and antiarrhythmic drugs. A, average dose-response curves for the enantiomers of D888 (devapamil) and verapamil. Data shown represent the mean ± S.E.M. from three to five experiments. The enantiomers of D888 were compared simultaneously in five experiments. Although the Hill coefficient (n_H) averaged ∼0.7, this was not statistically different from observed with verapamil (n_H = 1.0, data presented in Table 1). Structures and symbol key are given to the right of the graph. The structure for D888 corresponds to the (-)-(R)-enantiomer. Verapamil has an extra methoxy side group on one of its phenyl rings. B, dose-response analysis of perhexiline, amiodarone, and bepridil.

Fig. 3.

Stereoselectivity of block by dihydropyridines. A, block by the enantiomers of Bay K8644. Structure of (+)-(R)-Bay K8644 is shown to the right of the dose-response graph. Although the side chains in Bay K8644 are smaller than for most dihydropyridines tested, the (S)-enantiomer is an agonist at L-type channels, whereas the (R)-enantiomer is an antagonist. In contrast, these enantiomers showed a similar potency at blocking Ca_v3.2. B, enantiomers of efonidipine also showed a similar potency to block Ca_v3.2, and the block occurred at 10-fold lower concentrations than with Bay K8644. The racemic mixture showed a similar block as either enantiomer. The structure shown corresponds to the (-)-(R)-enantiomer. C, block by the enantiomers of niguldipine. Again, the structure shown corresponds to the (-)-(R)-enantiomer, highlighting that the large hydrophobic side chains of niguldipine occur on the opposite side of the dihydropyridine stereocenter compared with efonidipine. Block by these enantiomers was simultaneously compared with the racemic mixture in five experiments, and in each case, the (S)-enantiomer was ∼4-fold more potent.

Fig. 4.

Block of Ca_v3.2 by a series of dihydropyridines. A, dose-response curves of dihydropyridines that are capable of blocking with IC₅₀ values in the 3 to 6 μM range, which included felodipine, isradipine, nimodipine, nisoldipine, and nitrendipine. Because there were no statistical differences in the estimated potencies to block Ca_v3.2, only one fit to the data is shown, and the error bars were not plotted. Structures are shown to the right, except nimodipine, which is shown next to B. B, dose-response curves of dihydropyridines that block with IC₅₀ values in the 20 to 30 μM range (nifedipine and amlodipine) compared with a higher potency blocker (nicardipine). The lines represent fits to the average data, which are usually close to the average of fits to individual experiments. However, this was not the case for nifedipine and amlodipine, where the fit to the average produces a shallower slope (n_H = 0.5) than observed in individual experiments (Table 2).