Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

Abstract

We have previously defined in the NH 2 -terminal cytoplasmic domain of the mouse AE2/SLC4A2 anion exchanger a critical role for the highly conserved amino acids (aa) 336 -347 in determining wild-type pH sensitivity of anion transport. We have now engineered hexaAla ((A) 6 ) and individual amino acid substitutions to investigate the importance to pH-dependent regulation of AE2 activity of the larger surrounding region of aa 312-578. 4,4 -Diisothiocyanostilbene-2,2 -disulfonic acid (DIDS)-sensitive 36 Cl ؊ efflux from AE2-expressing Xenopus oocytes was monitored during changes in pH i or pH o in HEPES-buffered and in 5% CO 2 /HCO 3 ؊ -buffered conditions. Wild-type AE2-mediated 36 Cl ؊ efflux was profoundly inhibited at low pH o , with a pH o(50) value ‫؍‬ 6.75 ؎ 0.05 and was stimulated up to 10-fold by intracellular alkalinization. Individual mutation of several amino acid residues at non-contiguous sites preceding or following the conserved sequence aa 336 -347 attenuated pH i and/or pH o sensitivity of 36 Cl ؊ efflux. The largest attenuation of pH sensitivity occurred with the AE2 mutant (A) 6 357-362. This effect was phenocopied by AE2 H360E, suggesting a crucial role for His 360 . Homology modeling of the three-dimensional structure of the AE2 NH 2 -terminal cytoplasmic domain (based on the structure of the corresponding region of human AE1) predicts that those residues shown by mutagenesis to be functionally important define at least one localized surface region necessary for regulation of AE2 activity by pH.The anion exchangers AE1, AE2, and AE3 are polypeptide products of the SLC4 bicarbonate transporter gene superfamily that mediate Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchange. Anion exchangers contribute to the regulation of intracellular pH (pH i ), cell volume, tonicity, and intracellular Cl (Cl Ϫ ) in metazoan cells (1-5). The AE polypeptides share a highly conserved hydrophobic, polytopic, transmembrane domain of Ͼ500 amino acids (aa), 1 with a short COOH-terminal cytoplasmic tail capable of binding carbonic anhydrase II (6, 7). This transmembrane domain is preceded by a less extensively conserved hydrophilic NH 2 -terminal cytoplasmic domain of 400 -700 aa (3). The polytopic transmembrane domain can mediate anion exchange in the absence of nearly the entire NH 2 -terminal cytoplasmic domain (8, 9). Whereas the NH 2 -terminal cytoplasmic domain of erythroid AE1 is important for its binding to multiple cytoskeletal proteins, glycolytic enzymes, and hemoglobin (10), functions of the cytoplasmic NH 2 -terminal domains of AE2 and AE3 remain less extensively investigated.The polypeptide products of the various SLC4 AE anion exchanger genes differ in their acute regulation by pH. Native AE1-mediated Cl Ϫ /HCO 3 Ϫ exchange in erythrocytes (11) and heterologous AE1-mediated Cl Ϫ /Cl Ϫ exchange in Xenopus oocytes (12, 13) both display a broad pH versus activity profile, consistent with the primary role of erythroid AE1 in facilitating CO 2 /HCO 3 Ϫ exchange between the respiring tissues and lungs (14). In contras...

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