Identification and Functional Role of Calpain Cleavage Site in Na+-Ca2+ Exchanger 1 (NCX1)

Altered Ca2+ homeostasis is a key determinant of cardiac remodeling and contractility during chronic heart disease. Aberrant activation of calpain, a ubiquitous Ca2+-dependent protease can contribute to loss of Ca2+ control in cardiomyocytes. Calpain cleaves NCX1, but the underlying significance of the direct cleavage of NCX1 by calpain remains to be determined.By bioinformatics and mutational analysis we identified M369 as a putative calpain cleavage site residing within the α-catenin-like domain (CLD) in NCX1. Importantly, the cleavage of NCX1 at M369 corresponded to a proteolytic fragment of 75 kDa in left ventricular biopsies from aorta stenosis (AS) patients and in the failing left ventricle of rats following aortic banding (AB). Moreover, calpain binding in the CLD and Ca2+ -binding domain (CBD1) of NCX1 protein were identified by overlay assays. In order to investigate the specific function of M369 cleavage and avoid auxiliary and indirect functions of calpain, we utilized the protease Tobacco Etch Virus (TEV) to investigate site-specific cleavage. The TEV protease recognition site was inserted at the location of M369 in NCX1. By employing the patch clamp technique on transfected HEK cells, we observed a significant reduction of NCX1 current following TEV cleavage. In conclusion, we have identified and investigated the functional role of the calpain cleavage site at M369 in the CLD of NCX1. Our findings show that cleavage of the NCX1 at M369 reduces the total current. This reduction of NCX1 activity could potentially function to compensate for altered Ca2+ homeostasis associated with NCX upregulation during heart failure.