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Supriya S. Jalimarada, Eranga Vithana, Joseph Bonanno; Lack of Bicarbonate Transport by SLC4A11 in Bovine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5335.
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Bicarbonate transport is an integral part of the fluid transport/pump mechanism in corneal endothelium (CE). SLC4A11 is a member of the SLC4 superfamily of bicarbonate transporters and has been characterized as a putative sodium borate transporter. Using primary cultures of bovine corneal endothelial cells (BCEC), we ask if SLC4A11 functions as a Na-dependent bicarbonate transporter.
SLC4A11 expression in BCEC was knocked down using an SLC4A11 siRNA mixture. Knockdown was analyzed by western blot. Solute transport was assessed by measuring intracellular pH (pHi) and intracellular sodium concentration [Nai], using fluorescent dyes. Cells grown on coverslips were transfected with either scrambled-sequence or siSLC4A11 or treated with transfection reagent only and subjected to analysis 72 hrs post-transfection. Na and HCO3 transport was determined by examining pHi and [Nai] responses to CO2/HCO3 pulses in control and transfected cells. Additionally, effect of knockdown on Na/H+ exchange was assessed using Na free pulses on pHi.
siRNA transfection of BCEC resulted in ~70% knockdown of SLC4A11. Perfusion with CO2/HCO3 rich ringer induced a brief acidification (0.07 pH units) followed by alkalinization (0.2 pH units) in control cells. No significant difference in rates or amplitude of acidification and alkalinization was observed for the siSLC4A11 transfected cells, indicating lack of bicarbonate transport by SLC4A11. Addition of CO2/HCO3 induced a sharp increase in [Na]i. The initial rate of Na influx was not significantly different in siRNA treated cells. However, the amplitude of the [Na]i increase was 30% less (p=0.04) and the steady-state [Nai] was 46% lower (p= 0.005) in siSLC4A11 cells compared to control indicating decreased [Na]i. Na free pulses showed a 34% and 38% decrease in acidification in the absence and presence of EIPA (an inhibitor of Na+/H+ exchanger), respectively, compared to control.
Lack of changes in pHi to CO2/HCO3 in siSLC4A11 transfected cells indicates absence of any significant bicarbonate transport by SLC4A11. However, a decrease in sodium influx and lower steady state in SLC4A11 knockdown to CO2/HCO3, along with reduced Na free acidification & EIPA sensitivity suggests Na+/H+ exchanger like activity
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