Activation of the cardiac Na+–Ca2+ exchanger by sorcin via the interaction of the respective Ca2+-binding domains

Zamparelli, C., MacQuaide, N. , Colotti, G., Verzili, D., Seidler, T., Smith, G. L. and Chiancone, E. (2010) Activation of the cardiac Na+–Ca2+ exchanger by sorcin via the interaction of the respective Ca2+-binding domains. Journal of Molecular and Cellular Cardiology, 49(1), pp. 132-141. (doi:10.1016/j.yjmcc.2010.03.003) (PMID:20298697) (PMCID:PMC2958309)

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Abstract

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca2+ binding. The sarcolemmal Na+/Ca2+ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca2+ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca2+ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G > wt sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca2+, the interaction with CBD1 followed the order W105G > SCBD > wt sorcin > W99G > E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca2+-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Professor Godfrey and MacQuaide, Dr Niall
Authors: Zamparelli, C., MacQuaide, N., Colotti, G., Verzili, D., Seidler, T., Smith, G. L., and Chiancone, E.
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Journal of Molecular and Cellular Cardiology
Publisher:Elsevier
ISSN:0022-2828
Published Online:15 March 2010
First Published:First published in Journal of Molecular and Cellular Cardiology 49:132-141
Publisher Policy:Reproduced under a Creative Commons licence

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
378541A study of the subcellular actions of sorcin in ventricular cardiac muscleGodfrey SmithBritish Heart Foundation (BHF)PG/04/058/17157Institute of Cardiovascular and Medical Sciences