Jalimarada et al. (2013). Investigative Ophthalmology & Visual Science. doi: 10.1167/iovs.13-11929
…lin (100 U/mL)/fungizone (0.25 lg/mL) mixture in T-25 flasks at 378C with 5% CO2 until confluent, 5 to 7 days. Cells (3.5 3 105) were subcultured into six-well plates or six 25-mm coverslips for experiments, allowed to come to 100% confluence, and used within 24 to 48 hours. Rabbit eyes (Pel-Freez Biologicals, Roger, AR) were used to obtain corneas from which the endothelial layers from the poster
Abstract
<h4>Purpose</h4>Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na⁺ borate and Na⁺:OH⁻ transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]₄⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺.<h4>Methods</h4>SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na⁺ ([Na⁺](i)) in response to Ringer solutions with/without B(OH)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11.<h4>Results</h4>SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)₄⁻ (2.5-10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05-0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)₄⁻. However, the rate of B(OH)₄⁻-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)₄⁻ did not induce significant changes in resting [Na⁺(i)] or the amplitude and rate of acidification caused by Na⁺ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO₂/HCO₃⁻-rich Ringer, Na⁺-free induced acidification, or the rate of Na⁺ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na⁺ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH₄⁺ prepulse was decreased significantly (27%) by SLC4A11 silencing.<h4>Conclusions</h4>In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.