Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pH(i)) and HCO(3)(-) metabolism. Electrogenic NBC1 catalyzes HCO(3)(-) 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 CO(2)/HCO(3)(-). The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pH(i) in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pH(i) 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 pH(i) 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 pH(i) 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 HCO(3)(-) transport site maximizes the transmembrane HCO(3)(-) gradient local to NBC1 and thereby activates the transport rate.
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(NBC3Ct) with Kd ϭ 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 Ϯ 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 M 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH i) recovery rate by 31 Ϯ 3, or 38 Ϯ 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH i recovery rate by 69 Ϯ 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [␥-32 P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO 3 Ϫ transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.
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