Activation of TRPC6 channel proteins: evidence for an essential role of phosphorylation.

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In excitable and non-excitable cells, activation of receptor-operated Ca2+-permeable cation channels (ROCs) by hormones, growth factors and neurotransmitters has been implicated in many physiological processes, such as cell growth and proliferation, smooth muscle contraction and synaptic transmission. Little is known about the molecular identity of ROCs or how they are activated, and therefore discovery of the transient receptor potential (TRP) family of cation channel proteins has been an exciting breakthrough in the quest to answer these questions (Clapham et al. 2001). Expression studies have shown that the TRPC subgroup of proteins (TRPC1–7) have properties similar to ROCs in native tissues, with activation mechanisms often involving phospholipase C-mediated signalling cascades leading to production of diacylglycerol (DAG), which opens channels via a protein kinase C (PKC)-independent mechanism. In this issue of The Journal of Physiology, Shi et al. (2004) report a thorough investigation on the regulation by Ca2+ of two closely related TRPC channel proteins, the murine homologues of TRPC6 and TRPC7 proteins, expressed in HEK293 cells using a combination of molecular and patch clamp techniques. The paper provides compelling evidence that external ([Ca2+]o) and internal Ca2+ concentrations ([Ca2+]i) have multiple effects on TRPC6/7 activity and that a Ca2+–CaM-dependent phosphorylation process is essential for the activation of TRPC6 but not TRPC7. The authors show that [Ca2+]o has biphasic effects on TRPC6 currents, increasing current density at submillimolar concentrations and inhibiting at supra-millimolar levels whilst TRPC7 currents are only inhibited by increasing [Ca2+]o. Inhibition of TRPC6/7 activity by [Ca2+]o is thought to be due to channel block by Ca2+ ions whereas potentiation of TRPC6 may be due to effects of [Ca2+]o on unitary conductance and channel kinetics. Shi et al. (2004) also demonstrate that [Ca2+]i has profound effects on TRPC6/7 currents by enhancing and then attenuating their activity as [Ca2+]i is increased within the physiological range. The paper clearly shows that TRPC6 activity is attenuated by a calmodulin (CaM) antagonist, coexpression of a Ca2+-insensitive mutant form of CaM, a CaM kinase II inhibitor, a CaM kinase II inhibitory peptide and by substitution of internal ATP with its non-hydrolysable analogue AMP-PNP. The authors propose that a Ca2+–CaM-dependent phosphorylation process involving CaM kinase II (probably at the channel protein) is an essential prerequisite for TRPC6 channels to be activated by OAG, which is a novel discovery that may be replicated for the activation of other TRP proteins. In comparison TRPC7 activity was enhanced by inhibiting CaM and also by applying IP3 to the cytoplasmic surface, which was reversed by increasing [Ca2+]i or adding CaM. These data suggest that Ca2+–CaM and IP3 regulate activation of TRPC7 by a direct inhibitory effect on channel proteins, possibly by acting at a common binding domain. In contrast application of IP3 had no effect on TRPC6 channel activity. The inactivation of TRPC6/7 currents by higher concentration of [Ca2+]i was also studied and found to be prevented or enhanced by agents that inhibit and activate PKC, respectively, indicating the involvement of Ca2+-dependent PKC activity. In a previous paper the authors provided evidence that TRPC6 is an essential component of noradrenaline-evoked ROCs in portal vein myocytes (Inoue et al. 2001), and this is still one of only a few papers to correlate a TRP protein to a native conductance. The present work provides further evidence that expressed TRPC6 protein has properties similar to ROCs in portal vein, but it also suggests that some discrepancies exist. Aromolaran et al. (2000) also showed that a phosphorylation process is important for activating ROCs in portal vein, but in contrast to expressed TRPC6, the phosphorylation process had a pharmacological profile similar to myosin light chain kinase and not CaM kinase II. In addition a recent study has shown that IP3 acts synergistically with DAG to fully activate ROCs in portal vein (Albert & Large, 2003) whereas Shi et al. (2004) state that IP3 has no effect on expressed TRPC6 activity. The reasons for these differences between expressed TRPC6 proteins and TRPC6-like currents in portal vein are not known but Shi et al. suggest that they could be due to ROCs in portal vein being composed of different TRPC subunits including TRPC6. Whatever the outcome of the molecular identity of ROCs in portal vein myocytes, the present work indicates that phosphorylation by CaM kinase II of TRPC6 is an essential priming event before activation by DAG.