Acute regulation of mouse AE2 anion exchanger requires isoform-specific amino acid residues from most of the transmembrane domain

The widely expressed anion exchanger polypeptide AE2/SLC4A2 is acutely inhibited by acidic intracellular (pH i ), by acidic extracellular pH (pH o ), and by the calmodulin inhibitor, calmidazolium, whereas it is acutely activated by NH 4 + . The homologous erythroid/kidney AE1/SLC4A1 polypeptide is insensitive to these regulators. Each of these AE2 regulatory responses requires the presence of AE2's C-terminal transmembrane domain (TMD). We have now measured 36 Cl − efflux from Xenopus oocytes expressing bi- or tripartite AE2–AE1 chimeras to define TMD subregions in which AE2-specific sequences contribute to acute regulation. The chimeric AE polypeptides were all functional at pH o 7.4, with the sole exception of AE2 (1-920) /AE1 (613-811) /AE2 (1120-1237) . Reciprocal exchanges of the large third extracellular loops were without effect. AE2 regulation by pH i , pH o and NH 4 + was retained after substitution of C-terminal AE2 amino acids 1120–1237 (including the putative second re-entrant loop, two TM spans and the cytoplasmic tail) with the corresponding AE1 sequence. In contrast, the presence of this AE2 C-terminal sequence was both necessary and sufficient for inhibition by calmidazolium. All other tested TMD substitutions abolished AE2 pH i sensitivity, abolished or severely attenuated sensitivity to pH o and removed sensitivity to NH 4 + . Loss of AE2 pH i sensitivity was not rescued by co-expression of a complementary AE2 sequence within separate full-length chimeras or AE2 subdomains. Thus, normal regulation of AE2 by pH and other ligands requires AE2-specific sequence from most regions of the AE2 TMD, with the exceptions of the third extracellular loop and a short C-terminal sequence. We conclude that the individual TMD amino acid residues previously identified as influencing acute regulation of AE2 exert that influence within a regulatory structure requiring essential contributions from multiple regions of the AE2 TMD.