PMID- 12411514
OWN - NLM
STAT- MEDLINE
DCOM- 20030501
LR  - 20190513
IS  - 0022-3751 (Print)
IS  - 1469-7793 (Electronic)
IS  - 0022-3751 (Linking)
VI  - 544
IP  - 3
DP  - 2002 Nov 1
TI  - Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of 
      Asp(986), Asp(988) and kNBC1-carbonic anhydrase II binding.
PG  - 679-85
AB  - The HCO(3)(-) : Na(+) cotransport stoichiometry of the electrogenic sodium 
      bicarbonate cotransporter kNBC1 determines the reversal potential (E(rev)) and 
      thus the net direction of transport of these ions through the cotransporter. 
      Previously, we showed that phosphorylation of kNBC1-Ser(982) in the 
      carboxy-terminus of kNBC1 (kNBC1-Ct), by cAMP-protein kinase A (PKA), shifts the 
      stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the 
      cotransporter is electrostaticaly modulated. These results raise the possibility 
      that phosphorylated kNBC1-Ser(982), or other nearby negatively charged residues 
      shift the stoichiometry by blocking a bicarbonate-binding site. In the current 
      study, we examined the role of the negative charge on Ser(982)-phosphate and 
      three aspartate residues in a D986NDD custer in altering the stoichiometry of 
      kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in 
      an Ussing chamber for electrophysiological studies. Enhanced green fluorescence 
      protein (EGFP)-tagged mutant constructs expressed in the same cells were used to 
      determine the phosphorylation status of kNBC1-Ser(982). The data indicate that 
      both kNBC1-Asp(986) and kNBC1-Asp(988), but not kNBC1-Asp(989), are required for 
      the phosphorylation-induced shift in stoichiometry. A homologous motif (D887ADD) 
      in the carboxy-terminus of the anion exchanger AE1 binds to carbonic anhydrase II 
      (CAII). In isothermal titration calorimetry experiments, CAII was found to bind 
      to kNBC1-Ct with a K(D) of 160 +/- 10 nM. Acetazolamide inhibited the 
      short-circuit current through the cotransporter by 65 % when the latter operated 
      in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. 
      Acetazolamide did not affect the cotransport stoichiometry or the ability of 
      8-Br-cAMP to shift the stoichiometry. Although CAII does not affect the transport 
      stoichiometry, it may play an important role in enhancing the flux through the 
      transporter when kNBC1-Ser(982) is unphosphorylated.
FAU - Gross, Eitan
AU  - Gross E
AD  - Department of Urology, Case Western Reserve University, Cleveland, OH, USA. 
      ezg@po.cwru.edu
FAU - Pushkin, Alexander
AU  - Pushkin A
FAU - Abuladze, Natalia
AU  - Abuladze N
FAU - Fedotoff, Olga
AU  - Fedotoff O
FAU - Kurtz, Ira
AU  - Kurtz I
LA  - eng
GR  - R01 DK058563/DK/NIDDK NIH HHS/United States
GR  - T32 DK007789/DK/NIDDK NIH HHS/United States
GR  - DK07789/DK/NIDDK NIH HHS/United States
GR  - DK58563/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - J Physiol
JT  - The Journal of physiology
JID - 0266262
RN  - 0 (Sodium-Bicarbonate Symporters)
RN  - 30KYC7MIAI (Aspartic Acid)
RN  - EC 4.2.1.- (Carbonic Anhydrase II)
SB  - IM
MH  - Amino Acid Motifs/physiology
MH  - Animals
MH  - Aspartic Acid/*chemistry
MH  - Carbonic Anhydrase II/*metabolism
MH  - Cell Line
MH  - Electrochemistry
MH  - Mathematics
MH  - Mice
MH  - Phosphorylation
MH  - Protein Binding
MH  - Sodium-Bicarbonate Symporters/*chemistry/*metabolism
PMC - PMC2290621
EDAT- 2002/11/02 04:00
MHDA- 2003/05/02 05:00
PMCR- 2003/11/01
CRDT- 2002/11/02 04:00
PHST- 2002/11/02 04:00 [pubmed]
PHST- 2003/05/02 05:00 [medline]
PHST- 2002/11/02 04:00 [entrez]
PHST- 2003/11/01 00:00 [pmc-release]
AID - PHY_029777 [pii]
AID - 10.1113/jphysiol.2002.029777 [doi]
PST - ppublish
SO  - J Physiol. 2002 Nov 1;544(3):679-85. doi: 10.1113/jphysiol.2002.029777.