Ion transport function of SLC4A11 in corneal endothelium

Abstract

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]₄⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺.

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)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11.

Results: 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.

Conclusions: In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.

Keywords: SLC4A11; bicarbonate; borate transport; intracellular Na+; intracellular pH.

Figure 1

Expression of SLC4A11 in corneal endothelium. (A) Bovine PCR. (B) Rabbit PCR. Different sets of primers were used to check and confirm the mRNA expression of SLC4A11. (C) Western blot shows protein expression in human, rabbit, and bovine endothelium. Fresh tissue was obtained from dissected corneas by peeling the Descemet's membrane along with the corneal endothelium.

Figure 2

Immunofluorescence basolateral localization of SLC4A11. (A–C) Cultured BCEC, SLC4A11 (green), ZO-1 (red), and nuclei (blue); (D–F) fresh rabbit corneal endothelium, SLC4A11 (green), and nuclei (blue). (D) Inset, ×20.

Figure 3

Effect of borate on pH_i in BCEC overexpressing SLC4A11. (A) Addition of 2.5 or 10 mM borate induced slight acidification followed by alkalinization. Inset: Reversal on borate removal. (B) Average amplitude of acidification and alkalinization (2.5 and 10 mM, n = 12 and 7, respectively). (C) SLC4A11 expression detected with HA-tag antibody at different plasmid to lipofectamine 2000 concentration ratios (4:6 3:9, 4:10, 3:6, 2:5 μL), 48-hour posttransfection. (D) Membrane expression of the transfected SLC4A11 by surface biotinylation (total lysate [TL], unbound fraction [UB], and bound fraction [BD]). (E) Average rate (n = 5) of alkalinization in SLC4A11 overexpressing cells relative to control.

Figure 4

Lack of borate-dependent Na⁺ flux. (A) Cell acidification caused by Na⁺ removal in the presence and the absence of borate. All solutions had 0.25 μM EIPA. (B) Average rate. (C) Average amplitude of acidification in the presence and the absence of borate (n = 9).

Figure 5

Effect of borate on intracellular Na⁺ concentration. (A) No change in SBFI fluorescence ratio with 5 or 10 mM borate in control mock-transfected cells (n = 7); inset: change in SBFI fluorescence due to bicarbonate-rich (BR) perfusion. (B) No change in SBFI fluorescence ratio with 5 or 10 mM borate in SLC4A11 overexpressing cells (n = 7). Ouabain (100 μM), a sodium potassium ATPase inhibitor, was used as a positive control.

Figure 6

SLC4A11 knockdown using siRNA in BCEC. (A) Western blot of siSLC4A11-treated cells at different concentrations of siRNA. (B) Different time points at 30 nM siRNA. The percentage knockdown was compared with the protein levels in scrambled siRNA-treated cells, C, control. Approximately 70% knockdown was estimated compared with scramble siRNA in BCEC at 72 hours posttransfection.

Figure 7

Effect of SLC4A11 knockdown on HCO₃⁻ induced changes in pH_i. (A) Representative traces of pH_i for control (scrambled siRNA, n = 8). (B) siSLC4A11 (n = 14) treated BCEC. (C, D) The amplitudes and the initial rates of alkalinization, respectively, for siSLC4A11 compared with control.

Figure 8

Effect of SLC4A11 knockdown on HCO₃⁻ induced changes in intracellular Na⁺. (A) Representative traces depicting change in SBFI ratio (340/380) for control (scrambled siRNA, n = 8). (B) siSLC4A11 (n = 8) treated BCEC in response to BR. (C) Summary of initial rates of fluorescence ratio.

Figure 9

SLC4A11 knockdown has no effect on Na⁺-dependent HCO₃⁻ flux. (A) Representative traces of pH_i for Control (scrambled siRNA, n = 6). (B) siSLC4A11 (n = 8) treated BCEC when Na⁺ was removed in the presence of bicarbonate. (C, D) Summary of amplitude and rate of pH_i changes.

Figure 10

SLC4A11 knockdown slows Na⁺ free induced changes in pH_i in the absence of HCO₃⁻. (A) Representative trace of pH_i for control (scrambled siRNA, n = 7). (B) siSLC4A11 (n = 9) treated BCEC. (C–E) Summary bar graphs of rate and amplitude of acidification, and rate of recovery, respectively, in control and siSLC4A11-treated cells (*P < 0.05).

Figure 11

SLC4A11 knockdown slows recovery from acid load induced by an NH₄⁺ pulse under HCO₃⁻-free conditions. (A) Representative trace of pH_i for control (scrambled siRNA, n = 10). (B) siSLC4A11 (n = 14) treated BCEC. NH₄⁺, 20 mM ammonium chloride. (C) Summary bar graph of rate of recovery from acid load (*P = 0.0075).