Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pH(i)) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl(-)/Cl(-) and Cl(-)/HCO(3)(-) exchange are inhibited by acid pH(i) or extracellular pH (pH(o)). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl(-)/Cl(-) exchange, measured as DIDS-sensitive (36)Cl(-) efflux from Xenopus oocytes, was experimentally altered by varying pH(i) at constant pH(o) or varying pH(o). Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal (36)Cl(-) efflux at pH(o) 7.4 and acid shifted the pH(o) vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl(-) transport activity with retention of pH(o) sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH(o) sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pH(i), pH(i) sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH(i) and pH(o).