Mutation of SER766/770/771 inhibits acidosis-Erk-dependent activation of the Na+/H+ exchanger in the myocardium

Abstract

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates myocardial intracellular pH. Inhibition of NHE1 prevents hypertrophy and reduces ischemia–reperfusion (I/R) injury in animal models. Extracellular regulated kinase (Erk) mediated activation of NHE1 occurs during I/R of the myocardium. To understand regulation of NHE1 in the myocardium by phosphorylation we constructed adenoviruses which express wild type or mutant NHE1 (phosphorylation sites S766/770/771A). Additionally, wt and mutant NHE1 had mutations L163F/G174S, which increases NHE1 resistance to EMD87580 (NHE1 inhibitor) by 100-fold and allows measurement of exogenous NHE1 while inhibiting endogenous NHE1. Cardiomyocytes infected with wt NHE1 had increased NHE1 activity and further stimulation of exchanger activity by sustained intracellular acidosis (SIA). Stimulation was abolished when cells were treated with U0126 (Erk inhibitor). Levels of phosphorylated Erk 1/2 increased in cells treated with SIA, which was abolished by U0126. However, in cells infected with mutant NHE1, NHE1 activity was not stimulated by SIA. Immunoprecipitation of [32P]-labeled NHE1 from cardiomyocytes showed that SIA increases the level of phosphorylated wt NHE1 and this increase was blocked via treatment of U0126. In contrast, mutant NHE1 showed no increase in phosphorylation levels upon treatment with SIA, and basal levels of phosphorylation were low compared to wt NHE1. The results suggest that one or more of the amino acids Ser766/770/771 are important sites of phosphorylation by the ERK pathway and are essential for complete stimulation of NHE1 via SIA.