Research article summary (published 19 Jan 2004):

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

Full 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.

Author information

Author/s: Loiselle, Frederick B (FB); Morgan, Patricio E (PE); Alvarez, Bernardo V (BV); Casey, Joseph R (JR);

Affiliation: Canadian Institute of Health Research Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Canada T6G 2H7.

Journal and publication information

Publication Type: Journal Article; Research Support, Non-U.S. Gov't

Journal: American journal of physiology. Cell physiology (Am J Physiol Cell Physiol), published in United States. (Language: eng)

Reference: 2004-Jun; vol 286 (issue 6) : pp C1423-33

Dates: Created 2004/05/20; Completed 2004/07/14; Revised 2007/11/15;

PMID: 14736710, status: MEDLINE (last retrieval date: 2/18/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Acid-Base Equilibrium - drug effects; physiology Adenosine - analogs & derivatives; pharmacology Binding Sites - drug effects; physiology Carbonic Anhydrase II - genetics; metabolism Cell Line Cell Membrane - enzymology Cyclic AMP-Dependent Protein Kinases - genetics; metabolism Cytoplasm - drug effects; metabolism Epithelial Cells - enzymology

Epithelial Cells - enzymology Humans Mutation - genetics Phosphorylation - drug effects Protein Binding - drug effects; physiology Protein Structure, Tertiary - drug effects; physiology Protein Transport - drug effects; physiology Sodium-Bicarbonate Symporters - genetics; metabolism Transfection

Associated Chemicals: SLC4A7 protein, human (0) ; Sodium-Bicarbonate Symporters (0) ; 8-bromoadenosine (2946-39-6) ; Adenosine (58-61-7) ; Cyclic AMP-Dependent Protein Kinases (EC 2.7.11.11) ; Carbonic Anhydrase II (EC 4.2.1.-)

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