title: Molecular mechanism of kNBC1-carbonic anhydrase II interaction in proximal tubule cells.
abstract: 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, L(958)DDV and D(986)NDD 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.
authors.affiliation: Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. apushkin@mednet.ucla.edu
authors.forename: Alexander
authors.initials: A
authors.lastname: Pushkin
authors.forename: Natalia
authors.initials: N
authors.lastname: Abuladze
authors.forename: Eitan
authors.initials: E
authors.lastname: Gross
authors.forename: Debra
authors.initials: D
authors.lastname: Newman
authors.forename: Sergei
authors.initials: S
authors.lastname: Tatishchev
authors.forename: Ivan
authors.initials: I
authors.lastname: Lee
authors.forename: Olga
authors.initials: O
authors.lastname: Fedotoff
authors.forename: Galyna
authors.initials: G
authors.lastname: Bondar
authors.forename: Rustam
authors.initials: R
authors.lastname: Azimov
authors.forename: Matt
authors.initials: M
authors.lastname: Ngyuen
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: C498898:SLC4A4 protein, human; C498899:Slc4a4 protein, mouse; D027982:Sodium-Bicarbonate Symporters; D024402:Carbonic Anhydrase II