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Supriya S. Jalimarada, Diego G. Ogando, Eranga N. Vithana, Joseph A. Bonanno; Investigation of Ion Transport Function of NaBC1. Invest. Ophthalmol. Vis. Sci. 2011;52(14):407.
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Mutations in NaBC1 (SLC4A11), a transmembrane protein expressed in human corneal endothelium (CE), are associated with congenital hereditary and Fuchs corneal endothelial dystrophies but its function has not been established. Based on sequence homology with bicarbonate transporters (SLC4-) and a plant borate transporter (Bor1), NaBC1 is suggested to function as a sodium-dependent anion (borate/bicarbonate) transporter and one study has shown sodium borate transport activity. The current study tests the hypothesis that NaBC1 is a borate transporter in bovine corneal endothelial cells (BCEC).
Transport function of NaBC1 was assessed in control (endogenous) and transfected (overexpressing) BCEC by measuring changes in intracellular pH or cell volume by incubating cells grown on coverslips with BCECF-AM [2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester], a pH sensitive fluorescent dye or calcein AM (fluorescence quenching volume sensing) in presence of 2.5 and 10 mM borate in bicarbonate free ringer solution (BF, pH 7.5). The effect of borate on intracellular pH was also examined in the presence of DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt), an inhibitor of anion transporters. Additionally, sodium dependent borate transport was assessed by measuring the rate of cell acidification upon removal of sodium in the presence and absence of borate.
Exposing cells to borate produced a rapid but small acidification (~0.01 pH units, n=13) followed by a sustained alkalinization (+0.06, & +0.18 pH units with 2.5 and 10 mM borate (n= 12 & 7, respectively), which was not significantly changed by overexpression of NaBC1. The alkalinization was completely inhibited by 250 µM DIDS. The presence of borate did not change the sodium free induced rate of acidification. No changes in cell volume could be detected upon exposure to 2.5 or 10 mM borate in either transfected or control cells.
Initial acidification with borate is due to boric acid diffusion. Dose dependent alkalinization suggests cellular borate uptake and its inhibition by DIDS indicates facilitation by an anion transporter. However, overexpression of NaBC1 neither enhanced alkalinization nor induced any changes in cell volume. Lack of effect of borate on sodium free induced rate of acidification indicates that borate fluxes are not linked to sodium. These results suggest that NaBC1 is not a sodium dependent borate transporter.
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