Molecular mechanism of kNBC1–carbonic anhydrase II interaction in proximal tubule cells

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C‐terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1‐ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a...

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Bibliographic Details
Published in:The Journal of physiology 2004-08, Vol.559 (1), p.55-65
Main Authors: Pushkin, Alexander, Abuladze, Natalia, Gross, Eitan, Newman, Debra, Tatishchev, Sergei, Lee, Ivan, Fedotoff, Olga, Bondar, Galyna, Azimov, Rustam, Ngyuen, Matt, Kurtz, Ira
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Carbonic Anhydrase II - genetics
Carbonic Anhydrase II - metabolism
Carbonic Anhydrase II - physiology
Cells, Cultured
Humans
Kidney Tubules, Proximal - cytology
Kidney Tubules, Proximal - enzymology
Kidney Tubules, Proximal - metabolism
Mice
Molecular Sequence Data
Protein Binding - genetics
Research Papers
Sodium-Bicarbonate Symporters - genetics
Sodium-Bicarbonate Symporters - metabolism
Sodium-Bicarbonate Symporters - physiology
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Summary:We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C‐terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1‐ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C‐terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L958DDV and D986NDD in the wild‐type kNBC1‐ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1‐mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.
ISSN:0022-3751
1469-7793
DOI:10.1113/jphysiol.2004.065110