We recently reported that regulation of the murine Na⁺-independent Cl^– -HCO₃^– exchanger, AE2, by pH requires amino acid residues 312-347 of its N-terminal cytoplasmic domain (Stewart et al. 2001). Initial pH-sensitive data employing hexa-alanine bloc mutations across this region prompted closer study of individual alanine substitutions in AE2 amino acid residues 336-347. AE2 function was measured as DIDS-sensitive ³⁶Cl^– efflux from Xenopus oocytes during variation of extracellular pH (pH_o) with clamped or unclamped intracellular pH (pH_i), or during variation of pH_i at constant pH_o. Statistical significance was assessed by Student’s unpaired t test, where the level of significance was P < 0.05.

Wild-type (WT) AE2-mediated ³⁶Cl^– efflux was inhibited by acid pH_o, with a value of pH_o(50) = 6.87 ± 0.05 (± S.E.M., n = 36), and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the pre-equilibrated weak acid, butyrate (40 mM; n = 43). Systematic hexa-alanine [(A)₆] bloc substitutions between aa 312-347 revealed that hexa-bloc mutation A₍₆₎342-347 exhibited the greatest acid shift in pH_o(50) value of ~0.8 pH units (n = 10, P < 0.05). Two of the six (A)₆ mutants retained normal pH_i sensitivity of ³⁶Cl^– efflux, whereas the (A)₆ mutants 318-323 (n = 10), 336-341 (n = 9) and 342-347 (n = 10) were not stimulated by intracellular alkalinization (P < 0.05).

We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH_o and to pH_i were found independently and in concert. Interestingly, activation of AE2 E346A by intracellular alkalinisation remained intact (n = 25, P > 0.05), but was abolished by the mutation E346D (n = 9, P < 0.05). In contrast, E346A acid shifted the pH_o(50) value from WT AE2 by ~0.7 pH units (n = 12, P < 0.05).

Alanine substitution of conserved glutamate residues in the cytoplasmic N-terminal domain of two additional pH-sensitive polypeptides, cAE3 and the AE1_cyto/AE2_memb chimera, confirmed the importance of these residues in the regulation by pH of other AE anion exchangers. We hypothesize that individual amino acid residues of AE2 aa 336-347 contribute to a local structure that interacts, directly or indirectly, with pH-sensor residues of the AE2 transmembrane domain. This regulatory interaction is probably conserved among members of the SLC4 bicarbonate transporter gene superfamily.

We thank The Wellcome Trust for generous support.

All procedures accord with current National and local guidelines.