Stop Codon Polymorphisms in the Human SLC9A1 Gene Disrupt or Compromise Na+/H+ Exchanger Function.

The NHE1 isoform of the mammalian Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells by removing one intracellular proton in exchange for one extracellular sodium. Deletion of the NHE1 gene (SLC9A1) affects the growth and motor ability of mice and humans but mutations and polymorphisms of the gene are only beginning to be characterized. NHE1 has a cytosolic C-terminal regulatory tail of approximately 315 amino acids and a 500 amino acid membrane domain. We examined the functional effects of three human stop codon mutations at amino acids 321, 449 and 735 in comparison with a mutant that had a shortened tail region (543 stop codon). The short mutants, 321, 449 and 543 stop codon mutant proteins, lost NHE1 activity and expression, and did not target to the plasma membrane. Protein for these short mutants was more rapidly degraded than the wild type and 735 ending proteins. The 735 terminating mutant, with the membrane domain and much of the cytosolic tail, had reduced protein expression and activity. The results demonstrate that early stop codon polymorphisms have significant and deleterious effects on the activity of the SLC9A1 protein product. The 735-NHE1 mutant, without the last 80 amino acids, had more minor defects. Surprisingly, retention of a proximal 43 amino acids adjacent to the membrane domain did little to maintain NHE1 expression, targeting and activity.

Activation of the Na+/H+ exchanger in isolated cardiomyocytes through ß-Raf dependent pathways. Role of Thr653 of the cytosolic tail.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that is a key regulator of intracellular pH in isolated cardiomyocytes. A 500 amino acid membrane domain removes protons and is regulated by a 315 amino acid cytosolic domain. In the myocardium, aberrant regulation of NHE1 contributes to ischemia reperfusion damage and to heart hypertrophy. We examined mechanisms of regulation of NHE1 in the myocardium by endothelin and ß-Raf. Endothelin stimulated NHE1 activity and activated Erk-dependent pathways. Inhibition of ß-Raf reduced NHE1 activity and Erk-pathway activation. We demonstrated that myocardial ß-Raf binds to the C-terminal 182 amino acids of the NHE1 protein and that ß-Raf is associated with NHE1 in intact cardiomyocytes. NHE1 was phosphorylated in vivo and the protein kinase inhibitor sorafenib reduced NHE1 phosphorylation levels. Immunoprecipitates of ß-Raf from cardiomyocytes phosphorylated the C-terminal 182 amino acids of NHE1 and mass spectrometry analysis showed that amino acid Thr653 was phosphorylated. Mutation of this amino acid to Ala resulted in defective activity while mutation to Asp restored the activity. The results demonstrate that Thr653 is an important regulatory amino acid of NHE1 that is activated through ß-Raf dependent pathways by phosphorylation either directly or indirectly by ß-Raf, and this affects NHE1 activity.