Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex
| Autor: | Petra Eder, Sepp D. Kohlwein, Klaus Groschner, Heimo Wolinski, Christian Rosker, D Probst, Christoph Romanin, Michael Poteser |
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| Rok vydání: | 2007 |
| Předmět: |
Physiology
Biology Transfection Sodium-Calcium Exchanger Cell Line Rats Sprague-Dawley Transient receptor potential channel TRPC3 Physiology (medical) Animals Homeostasis Humans Immunoprecipitation Myocytes Cardiac Cells Cultured TRPC TRPC Cation Channels Calcium signaling Microscopy Confocal Phospholipase C Cell Membrane Sodium/Calcium Exchanger 1 Immunohistochemistry Molecular biology Rats Cell biology Type C Phospholipases Calcium ion homeostasis cardiovascular system Calcium Signal transduction Cardiology and Cardiovascular Medicine Signal Transduction |
| Zdroj: | Cardiovascular Research. 73:111-119 |
| ISSN: | 0008-6363 |
| DOI: | 10.1016/j.cardiores.2006.10.016 |
| Popis: | Objective: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca 2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na + /Ca 2+ 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. Coimmunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GSTpulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca 2+ entry, which was dependent on TRPC3-mediated Na + loading of myocytes. This NCX-mediated Ca 2+ 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 Ca 2+ homeostasis. |
| Databáze: | OpenAIRE |
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