title: Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA.
abstract: 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 left arrow over right arrow 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 microM 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 [gamma-(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.
authors.affiliation: Canadian Institute of Health Research Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Canada T6G 2H7.
authors.forename: Frederick B
authors.initials: FB
authors.lastname: Loiselle
authors.forename: Patricio E
authors.initials: PE
authors.lastname: Morgan
authors.forename: Bernardo V
authors.initials: BV
authors.lastname: Alvarez
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
mesh_terms: D000136:Acid-Base Equilibrium / Q000187:drug effects / Q000502:physiology*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology*; D006801:Humans; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C422022:SLC4A7 protein, human; D027982:Sodium-Bicarbonate Symporters; C036038:8-bromoadenosine; D017868:Cyclic AMP-Dependent Protein Kinases; D024402:Carbonic Anhydrase II; D000241:Adenosine