M1 muscarinic acetylcholine receptor regulation of endogenous transient receptor potential-canonical, subtype 6 (TRPC6) channels

In this study, we show that activation of M1 mAChRs in neuronal PC12D cells stimulates the formation of a multiprotein complex containing M1 mAChRs, TRPC6 channels and protein kinase C (PKC). TRPC6 channels in this complex are phosphorylated by PKC on a conserved serine residue in the carboxyl terminal domain. The immunophilin FKBP12 binds to the phosphorylated TRPC6 channel and creates a binding site for calcineurin/calmodulin, which dephosphorylates the channels. M1 mAChRs and PKC are transiently associated with TRPC6 channels by binding and dissociating within 5 min, while FKBP12, calcineurin, and calmodulin remain associated with the channels for at least 10 min. Dephophorylation of TRPC6 is required for the dissociation of M1 mAChRs, but PKC from the channels.; Prior to receptor activation, M1 mAChRs and TRPC6 channels localize to membrane domains that are soluble in the neutral detergent Triton X-100. Within 2 min after activation of the M1 mAChRs, the receptors and channels move into a detergent-resistant membrane (DRM) fraction that contains PKC and FKBP12. Phosphorylation of the channels by PKC and binding to FKBP12, calcineurin and calmodulin take place in the DRM fraction. The DRM fractionates at low-to-medium density in sucrose density gradients, which are enriched in caveolin-1, an integral membrane protein associated with caveolae. Within 5 min after activation, the M1 mAChRs undergo internalization via a clathrin-dependent pathway and the TRPC6 channels undergo internalization via a non-clathrin pathway. Both the M1 mAChRs and TRPC6 channels recycle to non-DRM fractions in the plasma membrane within 30 min after activation of M1 mAChRs. TRPC6 channels are activated in a non-DRM fraction of the plasma membrane and internalization of the TRPC6 channels functions to uncouple the channels from M1 mAChRs.; A TRPC6-centered multiprotein complex also forms in cultured primary hippocampal neurons following activation of endogenous muscarinic acetylcholine receptors. This complex contains the same components detected in PC12D cells: M1 mAChRs, TRPC6 channels, PKC, FKBP12, calcineurin and calmodulin. As observed in PC12D cells, the association of M1 AChRs and PKC with TRPC6 channels is transient, while the association of FKBP12, calcineurin and calmodulin with TRPC6 channels is stable for at least 10 min after exposure to carbachol.; Taken together, the results of this study provide new insights into mechanisms by which M1 mAChRs regulate TRPC6 channels. Future studies will build on this foundation to elucidate the molecular mechanisms underlying the activation and inactivation of the TRPC6 channels. The long-term goal of these studies is to elucidate the role of TRPC6 channels in M1 mAChR signaling in neurons and to determine the contribution of these channels to brain functions regulated by M1 mAChRs including memory and cognition.