PHOSPHORYLATION-DEPENDENT REGULATION OF CARDIAC NA+ CA2+ EXCHANGER VIA PROTEIN-KINASE-C/

The cardiac Na+/Ca2+ exchanger (NCX1) plays a major role in the extrus ion of Ca2+ from cardiomyocytes, We studied the role of protein phosph orylation in the regulation of cardiac NCX1 using CCL39 stably overexp ressing the canine cardiac NCX1 and rat neonatal cardiomyocytes, In bo th cell types, the NCX1 protein immunoprecipitated with a chicken anti -NCX1 antibody exhibited a significant basal phosphorylation that was further enhanced by treatment with endothelin-l, acidic fibroblast gro wth factor, phorbol 12-myristate 13-acetate, or okadaic acid, In contr ast, calphostin C, K252a, or EGTA inhibited the phosphorylation, The p hosphorylation occurred on two major tryptic phosphopeptides (Pi and P 2) exclusively on serine residues, Evidence is presented suggesting th at P2 was derived from an N-terminal half (amino acids 240-475) of the central cytoplasmic domain of NCX1 and was phosphorylated directly by protein kinase C (PKC), The agents that increased NCX1 phosphorylatio n significantly enhanced both the forward and reverse modes of Na+/Ca2 + exchange. This exchange activation exhibited a very good correlation with the NCX1 phosphorylation, In NCX1-transfected cells, PKC down-re gulation following prolonged exposure to phorbol 12-myristate 13-aceta te abolished the acidic fibroblast growth factor induced activation of exchange activity, On the other hand, cell ATP depletion reduced the exchange activity and abolished the effects of the above agents on exc hange activity, These results indicate that the cardiac NCX1 is upregu lated by PKC-catalyzed phosphorylation, The cardiac NCX1 thus could pl ay an important role in the previously reported negative inotropic act ions of phorbol esters and other PKC-activating agents.