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 (CaV3.1, CaV3.2, and CaV3.3) have not been systematically determined. The aim of the present study was to investigate whether there is 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 human embryonic kidney 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 that for CaV3.1 and CaV3.3. Block is 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, whereas 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.
Footnotes
This work was supported by the Consejo Nacional de Ciencia y Tecnología [Grant J50250Q] and the Instituto de Ciencia y Tecnología del Distrito Federal [Grant PICDS08−28] (to M.R.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- CaV
- voltage-gated calcium
- LVA
- low-voltage-activated
- HVA
- high-voltage-activated
- HP
- holding potential
- HEK
- human embryonic kidney
- AMI
- amiloride.
- Received March 24, 2012.
- Accepted July 5, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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