Putative Re-entrant Loop 1 of AE2 Transmembrane Domain Has a Major Role in Acute Regulation of Anion Exchange by pH

Abstract

Normal pH sensitivity of the SLC4A2/AE2 anion exchanger requires transmembrane domain (TMD) amino acid (aa) residues not conserved in the homologous but relatively pH-insensitive SLC4A1/AE1 polypeptide. We tested the hypothesis that the nonconserved aa cluster 1075 DKPK 1078 within the first putative re-entrant loop (RL1) of AE2 TMD contributes to pH sensor function by studying anion exchange function of AE2 mutants in which these and other RL1 aa were systematically substituted with corresponding RL1 aa from AE1. 1078 restored pH i sensitivity to the chimera AE2 (1-920) /AE1 (613-929) without affecting its low sensitivity to pH o . The results show that acute regulation of AE2 by pH requires RL1 of the TMD. We propose that critical segments of RL1 constitute part of an AE2 pH sensor that, together with residues within the N-terminal half of the TMD, constrain the AE2 polypeptide in a conformation required for regulation of anion exchange by pH i .The mouse SLC4A2/AE2 anion exchanger regulates intracellular pH (pH i ), 3 chloride concentration, cell volume, and transepithelial ion transport in many tissues and is essential to postweaning life in mice (1). SLC4A2/AE2 and its paralogs SLC4A1/AE1 and SLC4A3/AE3 each mediate Na ϩ -independent electroneutral anion exchange (2). The AE polypeptides are expressed in distinct tissue and cell type-specific patterns and exhibit distinct modes of acute regulation. AE2 and AE3 but not AE1 are acutely regulated by pH i (3). AE2 is also acutely regulated by extracellular pH (pH o ), NH 4 ϩ , hypertonicity, and (in a calmodulin-independent manner) calmidazolium (4, 5). Although the structural basis for these regulatory differences is not completely understood, recent structure-function analysis studies have identified isoform-specific residues in both N-terminal cytoplasmic and C-terminal transmembrane domains of AE2 that are required for acute regulation (4, 6).The 400 -700-aa N-terminal cytoplasmic domains of the SLC4 AE gene products are divergent in sequence, with only 35% aa identity between AE2 and AE1. The better conserved ϳ500-aa transmembrane domains (TMDs) of AE1, AE2, and AE3 share ϳ65% sequence identity. Even in the absence of their N-terminal cytoplasmic domains, AE TMDs expressed in erythrocytes, Xenopus oocytes, or mammalian cells can mediate electroneutral anion exchange (7-9). However, removal of all or part of the AE2 N-terminal cytoplasmic domain alters its acute regulation by pH, NH 4 ϩ , hypertonicity, and calmidazolium (5). Mutation of individual aa residues of the N-terminal cytoplasmic domain partially or completely recapitulates these regulatory changes. Moreover, many of these residues are predicted to reside on the surface of the N-terminal cytoplasmic domain (6). Removal of the N-terminal cytoplasmic domain from the AE2 TMD abolishes regulation by pH i (10) and significantly acid-shifts the pH o versus activity profile compared with wild-type AE2 (9). These findings suggest the presence of a pH sensor motif within the AE2 TMD that cooperates with se...

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