Cardiovasc Res. 2007 Jan 1;73(1):111-9. Epub 2006 Oct 26.
Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex.
Eder, P., Probst, D., Rosker, C., Poteser, M., Wolinski, H., Kohlwein, S. D., Romanin, C., Groschner, K.,
["Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria."]
["Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria."]
OBJECTIVE: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. METHODS: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. RESULTS: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. CONCLUSION: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.
PMID: 17129578

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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPC3 |
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NCX1 | Fusion protein-pull down assay | Human | 670-848 | E. coli | Not used | Rat Cardiomyocyte lysates | 17129578 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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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 | ||||
TRPC3 |
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NCX1 | Co-immunoprecipitation | Rat cardiomyocytes | 17129578 | |||||
TRPC3 |
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NCX1 | Co-immunofluorescence staining | Rat cardiomyocytes | 17129578 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC3 |
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NCX1 | Fusion protein-pull down assay | Human | 670-848 | Rat | Not determined | 17129578 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
