Regulation of the human NBC3 Na + /HCO 3 − cotransporter by carbonic anhydrase II and PKA

• Published in: American Journal of Physiology: Cell Physiology 2004-06, Vol.286 (6), p.C1423-C1433
• Main Authors: Loiselle, Frederick B., Morgan, Patricio E., Alvarez, Bernardo V., Casey, Joseph R.
• Format: Article
• Language: English
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00382.2003

Human NBC3 is an electroneutral Na + /HCO 3 − cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO 3 − metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO 2 + H 2 O ⇆ HCO 3 − + H + in many tissues. We investigated whether NBC3, like some Cl − /HCO 3 − 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 μM 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 pH i 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 [γ- 32 P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO 3 − transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.