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

Abstract

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 ⇆ 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 Kd= 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 μM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pHi) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHirecovery 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 [γ-32P]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.