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Department of Medicine, Harvard Medical School, Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Boston, MA 02215

Address correspondence to Seth L. Alper, RW763 East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Fax: (617) 667-8040; E-mail: salper{at}caregroup.harvard.edu

We reported recently that regulation by intracellular pH (pH_i) of the murine Cl^-/HCO₃^- exchanger AE2 requires amino acid residues 310–347 of the polypeptide's NH₂-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. ³⁶Cl^- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH_o) with unclamped or clamped pH_i, or during variation of pH_i at constant pH_o. Wild-type AE2–mediated ³⁶Cl^- efflux was profoundly inhibited by acid pH_o, with a value of pH_o(50) = 6.87 ± 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)₆]bloc substitutions between aa 312–347 identified the greatest acid shift in pH_o(50) value, 0.8 pH units in the mutant (A)₆342–347, but only a modest acid-shift in the mutant (A)₆336–341. Two of the six (A)₆ mutants retained normal pH_i sensitivity of ³⁶Cl^- efflux, whereas the (A)₆ mutants 318–323, 336–341, and 342–347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336–347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH_o and to pH_i were found independently and in concert. The E346A mutation acid-shifted the pH_o(50) value to the same extent whether pH_i was unclamped or held constant during variation of pH_o. Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH_o as well as pH_i.

Key Words: Cl^-/HCO₃^- exchange • weak acids • Xenopus oocytes • isotopic flux • pH-sensitive microelectrodes

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