Abstract

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_V3.1, Ca_V3.2, and Ca_V3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of Ca_V3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in Ca_V3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human Ca_V3 channels, we found that amiloride blocked the human Ca_V3 channels in a concentration-response manner; the IC₅₀ for Ca_V3.2 channels (62 μM) was 13-fold lower than that for Ca_V3.1 and Ca_V3.3. Block is voltage-independent (except for Ca_V3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in Ca_V3.1 channels. In addition, amiloride block of Ca_V3.2 channels is mainly due to an extracellular effect, whereas in Ca_V3.1 and Ca_V3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human Ca_V3 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_V3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.