School of Optometry, Indiana University, Bloomington, Indiana, USA.
Invest Ophthalmol Vis Sci. 2013 Jun 21;54(6):4330-40. doi: 10.1167/iovs.13-11929.
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⁺.
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⁺) in response to Ringer solutions with/without B(OH)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11.
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.
In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.
SLC4A11 是 SLC4 超家族碳酸氢盐转运体的成员，其突变导致角膜内皮细胞营养不良。SLC4A11 是一种假定的 Na⁺硼酸和 Na⁺：OH⁻转运体。因此，我们询问角膜内皮中的 SLC4A11 是否转运硼酸（B[OH]₄⁻）、碳酸氢盐（HCO3⁻）或羟基（OH⁻）阴离子与 Na⁺偶联。
通过半定量 PCR、SDS-PAGE/Western 印迹和免疫荧光染色来确定培养的原代牛角膜内皮细胞（BCEC）中 SLC4A11 的表达。在过表达或小干扰 RNA（siRNA）沉默 SLC4A11 后，通过测量细胞内 pH（pHi）或 Na⁺（[Na⁺]（i））来检测离子转运功能，以响应含有/不含 B（OH）₄⁻或 HCO₃⁻的 Ringer 溶液。
SLC4A11 在 BCEC 中定位于基底外侧膜。无碳酸氢盐的 Ringer 中的 B（OH）₄⁻（2.5-10 mM）诱导快速小酸化（0.01 pH 单位），随后碱化（0.05-0.1 pH 单位），与硼酸进入细胞后 B（OH）₄⁻扩散一致。然而，B（OH）₄⁻诱导的 pHi 变化的速率不受 SLC4A11 的过表达影响。B（OH）₄⁻不会引起静息 [Na⁺（i）]或 Na⁺去除引起的酸化幅度和速率的显著变化。siRNA 介导的 SLC4A11 敲低（约 70%）不会改变 CO₂/HCO₃⁻丰富的 Ringer 对 pHi 的反应、无 Na⁺诱导的酸化或存在碳酸氢盐时的 Na⁺内流速率。然而，在没有碳酸氢盐的情况下，siSLC4A11 敲低显著降低了由于 Na⁺去除和恢复（53%）引起的酸化的速率（43%）和幅度（48%）。此外，SLC4A11 沉默使 NH₄⁺预脉冲后的酸恢复速率显著降低（27%）。
在角膜内皮中，SLC4A11 显示出强大的 Na⁺偶联 OH⁻转运，但不转运 B（OH）₄⁻或 HCO₃⁻。
