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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 ([pH.sub.i]) and extracellular pH ([pH.sub.o]). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte [pH.sub.i]: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, N[H.sup.+.sub.4]. We find that both N[H.sub.2]-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the N[H.sub.2]-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 [Ca.sup.2+] ([Ca.sup.2+.sub.i]) diminished or abolished AE2 stimulation by N[H.sup.+.sub.4] and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of [Ca.sup.2+] signaling. Unlike AE2 stimulation by N[H.sup.+.sub.4] and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain. band 3; Xenopus oocyte; isotopic flux; site-directed mutagenesis; chloride-bicarbonate exchange; chloride-bicarbonate exchanger; intracellular calcium ion

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 (pH[sub i]) and extracellular pH (pH[sub o]). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pH[sub i]: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH[sub 4, sup +]. We find that both NH[sub 2]terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH[sub 2]-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 Ca[sup 2+] (Ca[sup 2+, sub i]) diminished or abolished AE2 stimulation by NH[sub 4, sup +] and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca[sup 2+] signaling. Unlike AE2 stimulation by NH[sub 4, sup +] and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain. [ABSTRACT FROM AUTHOR]