Molecular Mechanisms Underlying the Regulation of Sodium/Proton Exchanger Isoform 1 by Calcineurin B Homologous Protein 3.

摘要

Restoration of cardiac intracellular pH (pHi) following acidification is of crucial importance for maintenance of myocardial contractility. The major mechanism responsible for this is the function of the sodium/hydrogen exchanger isoform 1 (NHE1) which is the primary isoform of mammalian myocardium. However, elevated activity and expression of NHE1 is also detrimental in numerous diseases of the heart and studies have documented its role in cardiac hypertrophy and heart failure as well as exacerbation of myocardial injury during periods of ischemia/reperfusion. Furthermore, a vast amount of research over the past decade has demonstrated that inhibition of NHE1 by pharmacological antagonists attenuates both ischemic/reperfusion damage as well as hypertrophy. Regulation of the exchanger occurs primarily through its interaction with numerous proteins and biomolecules. One such novel partner is the Ca2+-binding protein CHP3/tescalcin, a member of the calcineurin B homologous protein family, which, unlike isoforms CHP1 and 2, is predominantly restricted to the heart in adult human tissue.;The CHP proteins have been shown to be N-myristoylated, and belong to the EF-hand superfamily of Ca2+-binding proteins. By mutating the N-myristoylation domain as well as the sole functioning EF-hand motif, both the upregulation of NHE1 activity was ablated along with the cell surface stability of the exchanger. We determined that although neither site is required for the interaction with NHE1 or for promoting the maturation of the exchanger, both are necessary to stabilize NHE1 at the cell surface, thereby optimizing its plasmalemmal expression and activity. Furthermore, our results suggest that CHP3 is a member of the Ca2+-myristoyl switch protein family since mutation of either motif by itself resulted in identical regulation of the exchanger, but mutation of both sites in concert does not compound the decrease in exchanger activity or stability.;NHE1 maintains a distinct distribution within the myocardium where it is localized predominantly to the intercalated disks and transverse t-tubules, but not the sarcolemmal membrane. However, upon low flow ischemia/reperfusion or depletion of cellular ATP, NHE1 rapidly redistributes to the lateral sarcolemmal membranes. Furthermore ATP-depletion in AP-1 cells expressing NHE1 results in a decrease in Na+/H+ exchange activity which correlates partially to a dephosphorylation and depletion of the plasma membrane phosphoinositide, phosphatidylinositol-4,5-bisphosphate. Our results demonstrate ATP-depletion also causes in a rapid decrease in NHE1 at the cell surface of exchanger expressing AP-1 cells that correlates with the rapid inhibition of exchange activity. Furthermore, CHP3 over-expression is unable to stabilize the exchanger at the cell surface during episodes of ATP-depletion.;By utilizing both in vitro and in vivo binding assays as well as confocal fluorescent microscopy in concert with mutational analysis of the regulatory C-terminal motif of NHE1, we determined that CHP3 binds the NHE1 at the juxtamembrane region of the exchanger that is identical to the region that interacts with the other CHP isoforms. Furthermore, functional analysis of the exchanger expressed in NHE-deficient Chinese hamster ovary AP-1 cells, determined that CHP3 upregulates NHE1 by accelerating both biosynthetic maturation and cell surface stability of the exchanger.