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

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca²⁺ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.     

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

The sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is an important regulator of intracellular Ca²⁺ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na⁺/K⁺-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca²⁺ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X_5–8Φ₁Φ₂-X_8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.     

Mapping the in vitro interactome of cardiac sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na⁺)‐calcium (Ca²⁺) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca²⁺ homeostasis, serving as the primary mechanism for Ca²⁺ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1_cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.