Calcium-dependent cleavage of the Na(+)/Ca(2+) exchanger by m-calpain in isolated endoplasmic reticulum.
We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na(+)/Ca(2+) exchanger (NCX) in the ER. Treatment of the ER with Ca(2+) (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 microM) or MDL28170 (10 microM), or Ca(2+) chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca(2+) overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.
Samanta
Krishna
K
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Kar
Pulak
P
Chakraborti
Tapati
T
Chakraborti
Sajal
S
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D004151:Dipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D011651:Pulmonary Artery / Q000166:cytology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D004151:Dipeptides; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C071482:calpeptin; D004533:Egtazic Acid; C033668:calpastatin; D002154:Calpain; C449000:m-calpain; D002118:Calcium; C058076:calpain inhibitor III