Structure-function relationships of AE2 regulation by Cai

Chernova, Marina N., Andrew K. Stewart, Lianwei Jiang, David J. Friedman, Yune Z. Kunes, and Seth L. Alper. Structure-function relationships of AE2 regulation by Cai 2 -sensitive stimulators NH4 and hypertonicity. Am J Physiol Cell Physiol 284: C1235–C1246, 2003. First published January 15, 2003; 10.1152/ajpcell.00522.2002.—We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pHi) and extracellular pH (pHo). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pHi: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH4 . We find that both NH2terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH2-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336–347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic Ca2 (Cai 2 ) diminished or abolished AE2 stimulation by NH4 and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca2 signaling. Unlike AE2 stimulation by NH4 and by hypertonicity, AE2 inhibition by calmidazolium required only AE2’s COOH-terminal transmembrane domain.