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 1075DKPK1078 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. Regulation of Cl–/Cl– and Cl–/HCO–3 exchange by intracellular pH (pHi) or extracellular pH (pHo) was measured as 4,4′-di-isothiocyanatostilbene-2,2′ disulfonic acid-sensitive 36Cl– efflux from Xenopus oocytes. AE2 RL1 mutants 1075AAAQ1078 and 1075AAAQN1079 showed reduced pHi sensitivity and pHo sensitivity was acid-shifted by ∼1 pH unit. Individual mutants D1075A and P1077A exhibited moderately altered pH sensitivity, whereas a range of substitutions at conserved AE2 Ile-1079 substantially altered sensitivity to pHo and/or pHi. Substitution of the complete AE1 RL1 with AE2 RL1 failed to confer AE2-like pH sensitivity onto AE1. Replacement, however, of AE1 RL1 763SGPGAAAQ770 with AE2 1071VAPGDKPK1078 restored pHi sensitivity to the chimera AE2(1–920)/AE1(613–929) without affecting its low sensitivity to pHo. 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 pHi.
An alternative method normalized each rate constant to that individual oocyte's rate constant measured at pHo 8.5, which was given a value of 100%. The normalized rate constants at every pHo value were fit with Equation 1. Data calculated by this approach yielded pHo(50) values not significantly different in most cases from those presented. However, application of this alternative method to Equation 1 yielded poor fits (r2 as low as 0.6) for some AE2 mutants.
This work was supported, in whole or in part, by National Institutes of Health Grants DK43495 (to S. L. A.) and RR017927 (Shared Instrument Grant) to Beth Israel Deaconess Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
