We have compared regulation by pH of AE1 (band 3)- and AE2-mediated Cl uptake into Xenopus oocytes. Cl influx was assayed at varying extracellular pH (pH) values between 9.0 and 5.0 under conditions in which corresponding intracellular pH (pH) values were at or near steady-state. Wild type (WT) AE1 displayed a broad convex pH versus activity curve, with peak activity at pH 7.0 and 63% of maximal activity at pH 5.0. In contrast, WT AE2 displayed a steep pH versus activity curve, with peak activity at pH9.0 and full suppression at pH 5.0. The structural basis of these differing pH sensitivities was examined by expression of cRNAs encoding chimeric and truncated proteins. Mutant polypeptides were expressed in oocytes and detected at the cell surface. The AE2/AE1 polypeptide displayed a broad pH versus activity curve similar to that of WT AE1. In contrast, the AE1/AE2 polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values from that of WT AE2 by 0.69 ± 0.04 pH units. Moreover, whereas the pH versus activity curves of AE2 99 and WT AE2 were indistinguishable, AE2 510 exhibited a pH versus activity curve acid-shifted from that of WT AE2 by 0.66 ± 0.13 pH units (indistinguishable from that of AE1/AE2). The data suggest that a pH sensor resides within the transmembrane region of AE2. The affinity for protons of this pH sensor is influenced by a modifier site located between residues 99 and 510 of the N-terminal cytoplasmic domain of AE2. Acidification of oocytes with acetate suggested that pH accounted for some but not all of the measured pH dependence of AE2.

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