Blocking of T-Type Calcium Channels by TTA-A2 Reveals a Conservative Binding Site for State-Dependent Antagonists

T-type calcium channels, or CaV3 channels, are involved in several cell functions, and they can generate low-threshold spikes that lead to burst firing and oscillatory behavior in various types of neurons. In addition, CaV3 channels are also implicated in a number of pathophysiological processes such as epilepsy, sleep disorders, hypertension, neuropathic pain and cancer. However, until recently there were no potent and selective antagonists of CaV3 channels that facilitate further investigation of T-type channel function in neurological diseases and to validate these channels as efficient drug targets. Recently, a series of potent and selective T-type calcium channel blockers (T-type antagonists or TTAs) have been reported in several studies. Here, we describe the identification of one aminoacid that is determinant for the molecular coupling of TTA-A2 ([2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]pyridin-2-yl}ethyl)acetamide]) on the CaV3.1 recombinant human T-type calcium channel. By using a combination of molecular modeling, docking studies, mutagenesis and patch-clamp experiments of CaV3.1 channels expressed in HEK-293 cells, we have collected experimental evidence that supports the existence of a conservative binding site for TTA-A2 and state-dependent antagonists similar to TTA-A2, e.g., mibefradil. Blocking potency of TTA-A2 was reduced by two thirds in CaV3.1 mutants, where the identified aminoacid was replaced for alanine, proline, or arginine, among others. Sequence alignment of the three human CaV3 channels suggest that the binding site is conserved in the CaV3 subfamily, but not in the L-type CaV1.2 channel. In addition, our data also provides further evidence that TTA-Q4, a positive allosteric modulator of TTA-A2 functional inhibition, does not share the same binding site with the latter. Our results contribute to the knowledge for the rational design of T-type channel antagonists based on the structure of the TTA-A2 binding site.Supported by CONACYT-MEXICO 167790.