J Biol Chem. 2005 Sep 9;280(36):32035-47. Epub 2005 Jun 30.

Activation of M1 muscarinic acetylcholine receptors stimulates the formation of a multiprotein complex centered on TRPC6 channels.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Kim, J. Y., Saffen, D.,
["Department of Pharmacology, College of Medicine and Public Health, the Ohio State University Columbus, Ohio 43210, USA."]
In this study we showed that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) activates endogenous transient receptor potential-canonical, subtype 6 (TRPC6), channels in neuronal PC12D cells. Activation of TRPC6 channels is correlated with the formation of a multiprotein complex containing M1 mAChRs, TRPC6 channels, and protein kinase C (PKC). Formation of the M1 mAChR-TRPC6-PKC complex is transient, with highest levels reached approximately 2 min after stimulation of M1 mAChRs. PKC in the complex phosphorylates TRPC6 on a conserved serine residue in the carboxyl-terminal domain (Ser768 in the TRPC6A isoform and Ser714 in the TRPC6B isoform). The immunophilin FKBP12, the phosphatase calcineurin, and Ca2+-binding protein calmodulin are also recruited to the M1 mAChR-TRPC6-PKC complex following activation of M1 mAChRs and remain stably associated with the TRPC6 channels after M1 mAChRs and PKC have disassociated. Binding of FKBP12, calcineurin, and calmodulin to TRPC6 channels is blocked by the following: 1) inhibition of PKC; 2) mutation of the PKC phosphorylation site (Ser(7168/714)) in the channels; or 3) pretreatment with FK506 or rapamycin, immunosuppressants that directly bind FKBP12. Inhibition of FKBP12 binding blocks the dephosphorylation of TRPC6 channels and the disassociation of M1 mAChRs, without affecting disassociation of PKC. The calcineurin inhibitor cyclosporin A also blocks the dephosphorylation of TRPC6 and prevents the disassociation of M1 mAChRs. Together, these results show that activated TRPC6 channels form the center of a dynamic multiprotein complex that includes PKC and calcineurin, which respectively phosphorylate and dephosphorylate the channels. Phosphorylation of the TRPC6 channels by PKC is required for the binding of FKBP12, which in turn is required for the binding of calcineurin and calmodulin. Subsequent dephosphorylation of the channels by calcineurin is required for the disassociation of M1 mAChRs.
PMID: 15994335External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Calcineurin Inference Prediction 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 FKBP12 Inference Prediction 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 M1 mAChR Inference Prediction 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PKCơ Inference Prediction 15994335
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Calcineurin Co-immunoprecipitation PC12D 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Calmodulin Co-immunoprecipitation PC12D 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 FKBP12 Co-immunoprecipitation PC12D 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 M1 mAChR Co-immunoprecipitation PC12D 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PKCơ Co-immunoprecipitation PC12D 15994335
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 M1 mAChR Calcium measurement Activation 15994335
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PKCơ In vitro PTM assay Phosphorylation (Ser-714) 15994335
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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