Na+-dependent HCO3- import by the slc4a10 gene product involves Cl- export.
The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.
Damkier
Helle Hasager
HH
Water and Salt Research Center, Aarhus University, DK-8000 Aarhus, Denmark; Department of Anatomy, Aarhus University, DK-8000 Aarhus, Denmark. Electronic address: hd@ana.au.dk.
Aalkjaer
Christian
C
Water and Salt Research Center, Aarhus University, DK-8000 Aarhus, Denmark; Department of Physiology and Biophysics, Aarhus University, DK-8000 Aarhus, Denmark.
Praetorius
Jeppe
J
Water and Salt Research Center, Aarhus University, DK-8000 Aarhus, Denmark; Department of Anatomy, Aarhus University, DK-8000 Aarhus, Denmark.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D005452:Fluoresceins; D005456:Fluorescent Dyes; C526208:Slc4a10 protein, mouse; D027982:Sodium-Bicarbonate Symporters; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid