TY - JOUR
T1 - Activation of the cardiac Na(+)-Ca(2+) exchanger by sorcin via the interaction of the respective Ca(2+)-binding domains
AU - Zamparelli, Carlotta
AU - Macquaide, Niall
AU - Colotti, Gianni
AU - Verzili, Daniela
AU - Seidler, Tim
AU - Smith, Godfrey L.
AU - Chiancone, Emilia
N1 - Copyright 2010 Elsevier Ltd. All rights reserved.
Acceptance from VoR EKT 10.06.20
PY - 2010/7/1
Y1 - 2010/7/1
N2 - Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca(2+) binding. The sarcolemmal Na(+)/Ca(2+) 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 Ca(2+) 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 Ca(2+) due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) 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 50microM Ca(2+), 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 Ca(2+)-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.
AB - Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca(2+) binding. The sarcolemmal Na(+)/Ca(2+) 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 Ca(2+) 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 Ca(2+) due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) 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 50microM Ca(2+), 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 Ca(2+)-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.
KW - Animals
KW - Calcium/metabolism
KW - EF Hand Motifs
KW - Male
KW - Myocardium/metabolism
KW - Myocytes, Cardiac/cytology
KW - Protein Structure, Secondary
KW - Rabbits
KW - Sarcolemma/metabolism
KW - Sodium-Calcium Exchanger/genetics
U2 - 10.1016/j.yjmcc.2010.03.003
DO - 10.1016/j.yjmcc.2010.03.003
M3 - Article
C2 - 20298697
VL - 49
SP - 132
EP - 141
JO - Journal of molecular and cellular cardiology
JF - Journal of molecular and cellular cardiology
SN - 0022-2828
IS - 1
ER -