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

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.

Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

“…Sorcin is a calcium-binding protein, and the calcium-binding domain contains eight ␣-helices (A to H) organized in five calcium-binding motifs (EF1-EF5). EF2 and EF3 are linked by helix D and have a high affinity with metal (27). It has been previously reported that an F112L mutant of sorcin displays a marked decreased in Ca 2ϩ affinity (28).…”

Sorcin Induces a Drug-Resistant Phenotype in Human Colorectal Cancer by Modulating Ca2+ Homeostasis

“…Indeed, neuronal cells exposed to oxygen and glucose deprivation activate the Na þ /Ca 2þ exchanger NCX1, which is known to be a target protein of sorcin (14), and this is correlated with accumulation of Ca 2þ into the ER, and prevention of ER stress and apoptosis (42). Furthermore, increased levels of Ca 2þ in the ER have also been correlated with MDR phenotypes in cancer cells (41,43).…”

“…Sorcin is a Ca 2þ sensor which regulates the activity of the ryanodine receptor RyRs, the Na þ /Ca 2þ exchanger NCX, and the voltage-dependent L-type Ca 2þ channel (12)(13)(14) and that, by the interaction with these target proteins, is involved both in regulating Ca 2þ homeostasis and in modulating excitation-contraction coupling in the heart (15,16). Some evidence suggests involvement of sorcin in the drug resistance shown by human malignancies.…”

Roles of Sorcin in Drug Resistance in Cancer: One Protein, Many Mechanisms, for a Novel Potential Anticancer Drug Target

“…Calcium homeostasis Regulation of Ca 2+ channels, pumps, exchangers [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Regulation of ER and cytosolic Ca 2+ concentration [4, Regulation of heart-muscle contraction [6][7][8][9][10][11][12][13][14][15][16][17][18]29,30] Cellular metabolism ER stress sensor, unfolded protein response regulation [20,22,[26][27][28]31] Regulation of mitosis, cytokinesis, cell cycle [17,22,26,27,32] Regulation of kinases [22,30,33,34] Regulation of glucose metabolism …”