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Structural and Functional Changes in the C‐terminal Regulatory Tail of the Na+/H+ Exchanger Mediate Phosphorylation Induced Regulation
Author(s) -
Li Xiuju,
Fliegel Larry
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.882.1
Subject(s) - intracellular ph , sodium–hydrogen antiporter , phosphorylation , intracellular , extracellular , mutant , gene isoform , trypsin , chemistry , mutation , cytoplasm , biochemistry , microbiology and biotechnology , biology , enzyme , gene , sodium , organic chemistry
The Na + /H + exchanger isoform 1 (NHE1) is a plasma membrane pH regulatory protein that removes one intracellular H+ in exchange for an extracellular Na+. NHE1 is inactive at alkaline intracellular pH but is active with decreasing pHi. The set point of the protein is altered by phosphorylation of the cytoplasmic tail. We examined the mechanism by which phosphorylation mediates NHE1 activation. In the full length protein, mutation of Ser 770 and Ser 771 to either Asp or Ala resulted in an inability to stimulate the protein by sustained intracellular acidosis. By using western blotting against the HA‐tagged full length NHE1 protein, we found that NHE1 proteins with the mutation S726/729D and S770/771A showed no difference in the digestion pattern or sensitivity to digestion compared with the wild type. However, the S770/771D mutant always appeared to be more sensitive to digestion with trypsin than controls. We examined the expressed C‐terminal regulatory region of NHE1 with and without phosphomimetic S770/771D mutations. These NHE1 proteins had varying sensitivity to trypsin digestion. This difference did not occur with phosphomimetic mutations at S703D, S726/729D. The results suggest that phosphorylation of NHE1 at Ser 770 and Ser 771 causes a significant changed in the conformation of the NHE1 regulatory tail that may affect NHE1 activity. Supported by Canadian Institutes of Health Research.