Store-operated Ca2+ influx in native vascular smooth muscle cells relies on interactions between PKCδ, PIP2 and TRPC1 channels.

Store-operated-Ca2+-entry (SOCE) is increasingly recognized as an important signalling pathway mediating smooth muscle cell (SMC) contraction, proliferation, migration and growth (Trebak et al. 2013). In both excitable and non-excitable cells, SOCE is mediated by depletion of endoplasmic/sarcoplasmic reticulum (SR) Ca2+ stores. Store depletion is sensed by stromal interaction molecule (STIM) proteins on the SR membrane that undergo a conformation change and bind to and activate Orai1 Ca2+ channels in the plasma membrane, facilitating Ca2+ entry (Yeung et al. 2020). In synthetic vascular SMC (VSMC), interactions between STIM and Orai1 are well characterized and are assumed to play key roles in migration and proliferation. However, in native contractile VSMC, SOCE is considered to be mediated by other store-operated channels, such as canonical transient receptor potential 1 (TRPC1) channels, independently of Orai1 (Baudel et al. 2020a). The mechanisms linking SR store depletion to activation of TRPC1 channels in VSMC are controversial, although there is evidence that TRPC1 opening relies on interactions with protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP2). Shi et al. (2017) reported that SOCE via TRPC1 channels may involve a STIM1-mediated Gαq/phospholipase C (PLC) β1 pathway that induces TRPC1 opening by regulating PKC and PIP2 interactions. A recent study also suggested that activation of TRPC1 channels in VSMC relies on a PKC-dependent phosphorylation of TRPC1 channels as a perquisite for PIP2-mediated channel opening (Baudel et al. 2020a). The exact mechanisms underlying these pathways are not well understood, in part because the identification of the PKC isoform that interacts with TRPC1 channels to facilitate channel opening by PIP2 is not known. This issue was addressed in a recent issue of The Journal of Physiology by Baudel et al. (2020b). In their study, SOCEwas instigated in VSMC from rat mesenteric arteries by passive depletion of SR by incubation with the high-affinity Ca2+ chelator, BAPTA. The resultant store-operated whole cell currents in VSMC were greatly reduced by Pico 145, a TRPC1/4/5 blocker, as well as an externally acting TRPC1 antibody (T1E3). Furthermore, store-operated currents in VSMCswere reduced by a pan-PKC isoform inhibitor (GF109203X), demonstrating the importance not only of TRPC1 channels, but also PKC with respect to mediating SOCE. Although the PKC family consists of 11 different isoforms of different structures and functions, previous investigators had surmised that the specific isoform regulating TRPC1 channels in VSMCs required diacylglycerol for its activation, andwas insensitive to Ca2+. This eliminated all PKC isoforms with the exception of PKC δ, ε, η and θ (Baudel et al. 2020a). The specific PKC isoforms expressed in VSMC were therefore investigated at both the tissue and cellular level using western blot analysis and immunocytochemistry, respectively. The experiments concluded that PKCδ was the dominant PKC isoform expressed in rat mesenteric arteries, whereas PKCε was much lower and neither PKCη, nor θ were detected). Dialysis of a PKCε peptide inhibitor in the patch pipette had no effect on store-operated currents. However, a PKCδ peptide inhibitor and a cell-permeable PKCδ inhibitor (δV1-TAT) greatly reduced the currents. In addition, store-operated currents were severely impaired in VSMC in which PKCδ was genetically ablated by incubating VSMC for 48 h with anti-sense morpholino oligomers against PKCδ. Baudel et al. (2020b) next posited that, if PKCδ interacted with TRPC1 channels as a result of SR Ca2+ store depletion, then depletion of the SR store should evoke a physical interaction between PKCδ and TRPC1 channels. This was confirmed with both co-immunoprecipitation and proximity-ligation-assays (PLA), both of which demonstrated that depletion of SR Ca2+ stores, either with BAPTA or TPEN (a low affinity Ca2+ chelator), induced an interaction between PKCδ and TRPC1 channels. Baudel et al. (2020b) then refined these data and performed PLA experiments with a mixture of anti-phosphorylated serine/threonine residues and anti-TRPC1 antibodies, showing that, upon SR store depletion with BAPTA, these serine/threonine residues were phosphorylated (as demonstrated by an increase in fluorescent puncta in the PLA assay). Importantly, this phosphorylation of TRPC1 at the serine/threonine residues was inhibited by incubating VSMCwith the PKCδ inhibitor δV1-TAT or by inhibition of PKCδ with morpholinos. The involvement of PIP2 in this process was demonstrated by first showing that SR Ca2+ depletion in VSMC with BAPTA resulted in an interaction between PIP2 and TRPC1 (as indicated by increased fluorescent puncta in PLA experiments). This interaction was reversed with either the PKCδ inhibitor δV1-TAT or by promotion of PIP2 depletion with wortmannin. Furthermore, VSMC store-operated currents could be induced by increasing PKC activity via bath application of the phorbol ester 12,13-dibutyrate and this could also be enhanced by the inclusion of diC8-PIP2. Once again, these PIP2 mediated enhancements of store-operate currents were reversed by both the PKCδ inhibitor δV1-TAT and the TRPC1 antibody T1E3. Finally, application of the α1-adrenoceptor agonist methoxamine significantly increased TRPC1 channel activity and this was reversed by the PKCδ inhibitor δV1-TAT, suggesting that the TRPC1PKCδ -PIP2 signalling pathway might be involved in α1-mediated vasoconstriction of mesenteric blood vessels. The data reported by Baudel et al. (2020b) strongly suggest that PKCδ is an essential regulator of TRPC1 channels in freshly isolated contractile VSMC, phosphorylating the channel to make it susceptible to opening in response to PIP2 during SR Ca2+ store depletion. Although their study has addressed the specific