Direct Extracellular Interaction between Carbonic Anhydrase IV and the Human NBC1 Sodium/Bicarbonate Co-Transporter

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Abstract
Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pHi) and HCO3- metabolism. Electrogenic NBC1 catalyzes HCO3- fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydration−dehydration of CO2/HCO3-. The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pHi in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pHi recovery by 44 ± 3%, as compared to NBC1-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAIV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAIV interaction. NBC1-mediated pHi recovery rate after acid load was inhibited by 40 ± 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAII competes with endogenous CAII for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full pHi recovery activity. We conclude that CAIV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAII and CAIV close to the NBC1 HCO3- transport site maximizes the transmembrane HCO3- gradient local to NBC1 and thereby activates the transport rate.

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