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

Voltage-dependent CaV1.2 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(V)1.2 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(V)1.2. Aorta rings and isolated VSMCobtained from wild type or smooth muscle-selective conditional Ca(V)1.2 knock-out (Ca(V)1.2(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(V)1.2(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(V)1.2 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(V)1.2 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.