Conclusions
In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)&sub4;⁻ or HCO&sub3;⁻.
Methods
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)&sub4;⁻ or HCO&sub3;⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11.
Purpose
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]&sub4;⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺.
Results
SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)&sub4;⁻ (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)&sub4;⁻. However, the rate of B(OH)&sub4;⁻-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)&sub4;⁻ 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 CO&sub2;/HCO&sub3;⁻-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 NH&sub4;⁺ prepulse was decreased significantly (27%) by SLC4A11 silencing. Conclusions: In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)&sub4;⁻ or HCO&sub3;⁻.