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 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.
Pushkin
Alexander
A
Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. apushkin@mednet.ucla.edu
Abuladze
Natalia
N
Gross
Eitan
E
Newman
Debra
D
Tatishchev
Sergei
S
Lee
Ivan
I
Fedotoff
Olga
O
Bondar
Galyna
G
Azimov
Rustam
R
Ngyuen
Matt
M
Kurtz
Ira
I
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
C498898:SLC4A4 protein, human; C498899:Slc4a4 protein, mouse; D027982:Sodium-Bicarbonate Symporters; D024402:Carbonic Anhydrase II