Orai1 and TRPC1 Proteins Co-localize with CaV1.2 Channels to Form a Signal Complex in Vascular Smooth Muscle Cells

Abstract

Voltage-dependent Ca_V1.2 L-type Ca²⁺ channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca²⁺ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with Ca_V1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and Ca_V1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional Ca_V1.2 knock-out (Ca_V1.2^KO) mice were used to study vascular contractility, intracellular Ca²⁺ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in Ca_V1.2^KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that Ca_V1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and Ca_V1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca²⁺ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.

Keywords: CaV1.2; CaV1.2 channel; Orai1; TRPC1; Vascular tone regulation; calcium; calcium release-activated calcium channel protein 1 (ORAI1); excitation-contraction coupling (E-C coupling); ion channel; vascular smooth muscle cells.