FASEB J. 2007 Feb;21(2):608-17. Epub 2007 Jan 3.
Regulation of capacitative calcium entries by alpha1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of alpha1-syntrophin.
Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Raymond, G., Cognard, C., Bourmeyster, N., Constantin, B.,
["Institut de Physiologie et Biologie Cellulaires, CNRS, UMR-6187, University of Poitiers, 86022 Poitiers, France."]
["Institut de Physiologie et Biologie Cellulaires, CNRS, UMR-6187, University of Poitiers, 86022 Poitiers, France."]
Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane-associated cytoskeleton protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch-activated currents and store-operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and alpha1-syntrophin in muscle cells. TRPC1 was also associated with alpha1-syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S-transferase (GST) pull-down assays showed that TRPC1 binds to the alpha1-syntrophin PDZ domain. Transfected recombinant alpha1-syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and alpha1-syntrophin that may anchor the store-operated channels to the dystrophin-associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.
PMID: 17202249

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPC1 |
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Dystrophin | Inference | Prediction | 17202249 | |||||
TRPC1 |
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ơ1-syntrophin | Inference | Prediction | 17202249 |
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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 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 | ||
TRPC1 |
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ơ1-syntrophin | Fusion protein-pull down assay | Not used | Mouse myotube lysates | Not specified | 75-170 | E. coli | 17202249 |
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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 | ||||
TRPC1 |
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Dystrophin | Co-immunoprecipitation | Mouse hind limb muscle | 17202249 | |||||
TRPC1 |
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Dystrophin | Co-immunoprecipitation | SolC57 | 17202249 | |||||
TRPC1 |
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Dystrophin | Co-immunofluorescence staining | SolC57 | 17202249 | |||||
TRPC1 |
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ơ1-syntrophin | Co-immunoprecipitation | Mouse muscle lysates | 17202249 | |||||
TRPC1 |
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ơ1-syntrophin | Co-immunoprecipitation | SolC1 | Not used | Mouse | Full-length | 17202249 | ||
TRPC1 |
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ơ1-syntrophin | Co-immunoprecipitation | SolD6 | Not used | Mouse | Full-length | 17202249 |
(
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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 | ||||
TRPC1 |
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ơ1-syntrophin | Fusion protein-pull down assay | Mouse | Not determined | Not specified | 75-170 | 17202249 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
