Abstract

T-type calcium channels (T/Ca_v3 channels) are implicated in various physiological and patho-physiological 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 electrophysiological and pharmacological analysis indicating that the effects of the major N-acyl derivatives on the Ca_v3.3 current share many similarities with those of TTA-A2, a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [³H]-TTA-A1, we demonstrated that poly-unsaturated lipids which inhibit the Ca_v3.3 current, as NAGly, NASer, anandamide, NADA, NATau and NA-5HT, all displaced [³H]-TTA-A1 binding to membranes prepared from cells expressing Ca_v3.3, with K_i in a micromolar or sub-micromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca_v3.3 current, had no effect on [³H]-TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca_v3.3 current. In addition, TTA-Q4, a positive allosteric modulator of [³H]-TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca_v3.3 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 pharmacological tools to dissect the involvement of T-current in the physiological effects of endogenous lipids.