Abstract
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.
Highlights
NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na؉-dependent pHi recovery, which was accompanied by an increase in the intracellular Na؉ concentration evaluated by use of the Na؉-sensitive fluorophore CoroNa Green
Cl؊ is most likely the counterion maintaining electroneutrality because (i) Na؉-dependent pHi 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 HCO3؊-dependent 36Cl؊-efflux during pHi recovery was significantly greater in acidified slc4a10transfected cells than in control cells
The transport mode of the slc4a10 gene product has been investigated by more research groups defining the protein as an electroneutral Naϩ and HCO3Ϫ-dependent transporter which is inhibited by 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonate (DIDS) [2, 6, 7]
Summary
CDNA Constructs—The coding sequences of rat slc4a10 (rb2Ncbe, NBCn2-D, AY579374) and mouse slc4a10 (corresponding to rb1Ncbe, NBCn2-B, AB033759) were obtained by reverse transcription-PCR as described previously [9] and inserted into pcDNA5/FRT vectors (flipase recognition target; Invitrogen). Fluorophores—For pHi measurements, cells were loaded in 2 M BCECF-AM in a HEPES-buffered salt solution (HBS; Table 1) for 15 min. For intracellular [Naϩ] measurements, cells were loaded in 10 M CoroNa Green sodium indicator for 30 min in HBS. Cells were acidified for 3 min by adding NH4Cl to the solution (final concentration 20 mM) and washed four times in a Naϩ-free CO2/ HCO3Ϫ-buffered salt solution. The cells were incubated with Naϩ-containing BBS with the same ClϪ channel blocker. To compare values between wells of varying cell density, we normalized the counts for the background ClϪ in the final wash in Naϩ-free BBS. ClϪ influx was measured by exposing cells to 8 Ci/ml Na36Cl for 2 min corresponding to the initial period of Naϩ-dependent pHi recovery after NH4Cl prepulsing in the presence of 1 mM furosemide. Values of p Ͻ 0.05 were considered an appropriate level of statistical significance
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