RT Journal Article SR Electronic T1 Block of Human CaV3 Channels by the Diuretic Amiloride JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.112.078923 DO 10.1124/mol.112.078923 A1 Osbaldo Lopez-Charcas A1 Manuel Rivera A1 Juan C Gomora YR 2012 UL http://molpharm.aspetjournals.org/content/early/2012/07/05/mol.112.078923.abstract AB Previous studies in native T-type currents have suggested the existence of distinct isoforms with dissimilar pharmacology. Amiloride was the first organic blocker to selectively block the native T-type calcium channel, but the potency and mechanism of block of this drug on the three recombinant T-type calcium channels (CaV3.1, CaV3.2 and CaV3.3) have not been systematically determined. The aim of the present study was to investigate whether there is a differential block of CaV3 channels by amiloride, to establish the mechanism of block and to obtain insights into the amiloride putative binding sites in CaV3 channels. By performing whole-cell patch-clamp recordings of HEK-293 cells stably expressing human CaV3 channels, we found that amiloride blocked the human CaV3 channels in a concentration-response manner; the IC50 for CaV3.2 channels (62 μM) was 13-fold lower than for CaV3.1 and CaV3.3. Block was voltage-independent (except for CaV3.3 channels), and targets mainly closed-state channels, although a small use-dependent component was observed in CaV3.1 channels. In addition, amiloride block of CaV3.2 channels is mainly due to an extracellular effect; while in CaV3.1 and CaV3.3 channels the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human CaV3 channels differentially through a mechanism involving mainly the closed state of the channel, and suggest a negative allosteric interaction with at least two putative binding sites with different affinities. The preferential block of CaV3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.