Block of human CaV3 channels by the diuretic amiloride

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 (Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of Ca(V)3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in Ca(V)3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human Ca(V)3 channels, we found that amiloride blocked the human Ca(V)3 channels in a concentration-response manner; the IC₅₀ for Ca(V)3.2 channels (62 μM) was 13-fold lower than that for Ca(V)3.1 and Ca(V)3.3. Block is voltage-independent (except for Ca(V)3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in Ca(V)3.1 channels. In addition, amiloride block of Ca(V)3.2 channels is mainly due to an extracellular effect, whereas in Ca(V)3.1 and Ca(V)3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human Ca(V)3 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 Ca(V)3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.