Direct Fusion of the CAII Carbonic Anhydrase to the AE1 Cl‐/HCO3‐ Exchanger

Abstract

Some bicarbonate transporters bind to carbonic anhydrases (CA) to enhance their transport activities. Carbonic anhydrases catalyze the reversible conversion of CO2 to HCO3− + H+ and thus produce or consume the substrate of HCO3− transport. To further understand the physiological role of the CA/bicarbonate transporter interaction, we investigated the transport activity of the human Cl−/HCO3− exchanger isoform, AE1, by fusing CAII to its C-terminal region (AE1.CAII). As a control, AE1 was also fused to the catalytically inactive CAII mutant, V143Y, to generate AE1.CAIIV143Y. AE1 alone and the two fused proteins were expressed by transient transfection of HEK293 cells. Anion exchange activity, monitored from the rates of intracellular pH (pHi) change upon shifting extracellular Cl− from 140 mM to 0 mM and back, was determined by measuring the fluorescence of the pH indicator dye, 2′,7′-bis (2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF). The anion transport activity of AE1.CAII, normalized for AE1 expression level, was 119% ± 15% of cells transfected with AE1 alone. Interestingly, the transport activity of AE1.CAIIV143Y was markedly reduced (47% ± 10%) compared to AE1 alone. The reduction of activity of the AE1.CAIIV143Y fusion relative to AE1 alone suggests that the fusion compromises AE1 folding or activity. We conclude that catalytically active CAII tethered to the cytosolic surface of AE1 activates Cl−/HCO3−exchange activity.