Cross-Modulation and Molecular Interaction at the Cav3.3 Protein between the Endogenous Lipids and the T-Type Calcium Channel Antagonist TTA-A2.

T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the ω3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy)pyridin-2-yl)ethyl)acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [(3)H]TTA-A1 [(R)-2-(4-(tert-butyl)phenyl)-N-(1-(5-methoxypyridin-2-yl)ethyl)acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-l-serine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [(3)H]TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with Ki in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [(3)H]TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl)benzonitrile], a positive allosteric modulator of [(3)H]TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca(v)3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.