Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA

Human NBC3 is an electroneutral Na+/ HCO3- cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO3- metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO2 + H2O reversible arrow HCO3- + H+ in many tissues. We investigated whether NBC3, like some Cl- HCO3- exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K-d = 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 +/- 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 muM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH(i)) recovery rate by 31 +/- 3, or 38 +/- 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHi recovery rate by 69 +/- 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [gamma-P-32] ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO3- transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.