Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Abstract

Background: Sodium-calcium exchanger 1 (NCX1) and calpain are up-regulated in heart failure (HF). Molecular mechanisms and functional consequences of NCX1 cleavage by calpain are not known. Results: Calpain anchors to two NCX1 regions and cleaves at methionine-369, leading to inactivation. Conclusion: NCX1 inhibition by calpain might improve cardiac function. Significance: Calpain might play a pivotal role in NCX1 regulation during HF.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

“…11A). We have previously shown that the NCX1 level is up-regulated in response to pressure overload in our animal model (15), similar to what others have shown (5,50). Anti-calsequestrin and anti-total PLM were used as controls.…”

“…Staining without primary antibodies and use of an anti-NCX1 blocking peptide was used as a negative control. The proximity ligation assay was performed using the Duolink kit (DUO92014, Sigma) according to the manufacturer's protocol, as described previously (15). The cells were then incubated with 600 nM SYTOX Orange (S-11368, Life Technologies, Inc.), a nucleic acid stain, for 10 min at room temperature and rinsed three times for 5 min with PBS.…”

“…Recently, we have identified a calpain cleavage site at Met-369 in NCX1, which resides between the PLM-anchoring site (16) and PP1c-anchoring site (this study). Wanichawan et al (15) showed that calpain activity was greatly increased in ABHF hearts and that this correlated with increased cleavage of NCX1. To test whether calpain cleavage is responsible for dissociating PP1c away from PLM, HEK293 cells were co-transfected with PLM, FLAG-His 6 -PP1c(1-330), and NCX1(TEV).…”

“…The eukaryotic exchanger is composed of 10 transmembrane domains (TM) (11,12), with a large cytosolic loop between TM5 and TM6. Deletion of the cytosolic loop has revealed that it does not play a direct role in ion translocation, but rather it mediates regulation of the exchanger by associating with various cytosolic factors (13)(14)(15)(16). Relevant to this study are the interaction sites for phospholemman (PLM), a membrane phosphoprotein (14,16), and calpain, a non-lysosomal cysteine protease (15).…”

“…Deletion of the cytosolic loop has revealed that it does not play a direct role in ion translocation, but rather it mediates regulation of the exchanger by associating with various cytosolic factors (13)(14)(15)(16). Relevant to this study are the interaction sites for phospholemman (PLM), a membrane phosphoprotein (14,16), and calpain, a non-lysosomal cysteine protease (15). * This work was supported in part by the South-Eastern Norway Regional NCX1 has been shown to exist in a macromolecular complex comprising protein kinase A (PKA) and C (PKC), protein phosphatase 1 (PP1) and 2A (PP2A), as well as other anchoring and adaptor proteins (17).…”