The mammalian
Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory
membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after
ischemia-reperfusion injury and is implicated in
heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different
amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser(703), Ser(770), and Ser(771). In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular
acidosis. In this study, we determined whether Ser(703) and
p90(rsk) activity are critical in activation of NHE1 by sustained intracellular
acidosis. In vitro phosphorylation of NHE1 C-terminal fusion
proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser(703); however, phosphorylation by
p90(rsk) required Ser(703). A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular
acidosis in CHO cells. In addition, sustained intracellular
acidosis also activated the Ser703Ala
mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by
acidosis. The presence of a dominant-negative
p90(rsk)
kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular
acidosis in isolated cardiomyocytes. We conclude that Ser(703) and
p90(rsk) are not required for activation by sustained intracellular
acidosis and that
p90(rsk) phosphorylation of Ser(703) is independent of this type of activation.