Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies

Abstract

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na⁺ independent. Studies in oocytes and HEK293 cells could not detect Na⁺-coupled HCO₃^- transport or Cl^-/HCO₃^- exchange by SLC4A11. SLC4A11 mediated electroneutral NH₃ transport in oocytes. Voltage-dependent OH^- or H⁺ movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH^-/H⁺ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH₃, not NH₃/H.

Keywords: SLC4A11; ammonia; corneal dystrophy; endothelial cell; water flux.

Fig. 1.

SLC4A11 does not transport borate. A: cRNA-encoding N-terminally HA-epitope tagged SLC4A11 or water-injected X. laevis oocytes were incubated for 72 h. Oocytes were fractionated to isolate the membrane-rich fractions, and the samples were processed for immunoblotting with anti-HA antibody. B: borate transport was measured in oocytes by using an assay reported earlier (41). When borate enters the oocyte, osmolarity increases and water crosses the oocyte membrane to maintain transmembrane isoosmolarity. SLC4A11 or NIP5;1 cRNA or water-injected oocytes were incubated for 72 h and exposed to oocyte Ringer's (OR, white bar) solution (220 mosM/kg) for 1 min. Oocytes were then switched to OR isotonic solution containing 180 mM boric acid (black bar), while continuously monitoring under microscope and capturing images digitally every 15 s. Volume change was calculated by normalizing to average volume during initial 60 s. C: rate of volume change was represented by calculating the slope of initial swelling after switching to 180 mM borate solution. D: SLC4A11 cRNA-injected or native oocytes were incubated for 72 h and current was measured with a single-barreled microelectrode while perfusing the oocytes with oocyte Ringer's solution (OR 7.4, white bar), OR containing 1 mM boric acid (1 mM BA, black bar), and OR containing 5 mM boric acid (5 mM BA, black bar). E: SLC4A11-mediated membrane current, induced by switching to 1 mM or 5 mM boric acid is shown for native oocytes or SLC4A11 expressing oocytes. Error bars represent means ± SE (n = 7–10); *significant difference from H₂O injected oocytes (P < 0.05); n.s., not significant.

Fig. 2.

SLC4A11 mediates osmotic (independent of Na⁺) and electroneutral water movement in oocytes. A and B: oocytes (injected with cRNA-encoding N-terminally HA-epitope tagged SLC4A11 or AQP1 cRNA) were perfused with 220 mosM/kg isotonic oocyte ND96 medium (white bar), followed by 44 mosM/kg hypotonic solution (black bar). Perfusions proceeded in the absence (B) (replaced by choline chloride) or presence (A) of 96 mM NaCl. C: oocyte volume was calculated from images captured digitally every 15 s. Rate of volume change was represented by calculating the slope of initial swelling after switching to hypotonic solution. D: whole cell current was measured, with a single-barreled microelectrode, in oocytes injected with SLC4A11 cRNA or native oocytes. Oocytes were perfused with oocyte Ringer's solution (OR 7.4, white bar), 220 mosM/kg solution (isotonic, black bar), and 44 mosM/kg solution (hypotonic, white bar). E: SLC4A11 mediated membrane current, induced by switching from isotonic to hypotonic medium was determined for oocytes, during the first switch to hypo-osmotic medium. Error bars represent means ± SE (n = 7–10); *significant difference from H₂O-injected oocytes; #significant difference from SLC4A11-injected oocytes (P < 0.05); n.s., not significant.

Fig. 3.

Assessment of SLC4A11-mediated Na⁺ dependent or independent H⁺/OH⁻/HCO₃⁻ pH change in oocytes. SLC4A11 cRNA-injected or native oocytes were incubated for 72 h. pH and current were simultaneously measured with a double-barreled microelectrode combined with a two-electrode voltage clamp (clamped to −40 mV). A and B: cytosolic pH was measured as native (A) or SLC4A11-expressing (B) oocytes and were sequentially superfused with oocyte Ringer's solution containing 82.5 mM NaCl (OR 7.4, white bar), oocyte Ringer's solution containing only 5.85 mM NaCl (total [Na⁺] 10% of OR), and 2.4 mM HCO₃⁻, adjusted to pH 6.4 (10% Na⁺ HCO₃⁻, 6.4, black bar). Bicarbonate and bicarbonate-free oocyte Ringer's solutions were bubbled continuously with 5% CO₂-95% O₂. Arrows across the top bar indicate the time period when rates of pH change were measured, and the corresponding letters indicate the panel with the rates compared. C: Na⁺ dependent H⁺/OH⁻ ion movement mediated by SLC4A11 was calculated from rate of change of intracellular [H⁺] when oocytes were switched from OR 7.4 to 10% Na⁺, pH 6.4. D and E: Na⁺ independent or dependent bicarbonate movement mediated by SLC4A11 was calculated from rate of change in [H⁺]_I concentration when oocytes were switched from OR 7.4 to HCO₃⁻ 7.4 and then to 10% Na⁺ and HCO₃⁻, pH 6.4. Error bars represent means ± SE (n = 7).

Fig. 4.

SLC4A11 does not exhibit current changes during Na⁺ dependent or independent H⁺/OH⁻/HCO₃⁻ changes in oocytes. SLC4A11 cRNA-injected or native oocytes were incubated for 72 h. pH and current were simultaneously measured with a double-barreled microelectrode combined with a two-electrode voltage clamp (clamped to −40 mV). A and B: current was measured when the native or SLC4A11-expressing oocytes, and were exposed to oocyte Ringer's solution (OR 7.4, white bar), OR containing only 10% Na⁺, adjusted to pH of 6.4 (10% Na⁺ 6.4, black bar), OR containing bicarbonate (HCO₃⁻ 7.4, gray bar), and OR containing only 10% Na⁺ and HCO₃⁻, adjusted to pH of 6.4 (10% Na⁺ HCO₃⁻ 6.4, black bar). Arrows across the top bar indicate the time period when rates of [H⁺]_i were measured, and the corresponding letters indicate the panel with the rates compared. C: membrane current mediated by SLC4A11 was calculated when oocytes were switched from OR 7.4 to 10% Na⁺, pH 6.4. D and E: membrane current mediated by SLC4A11 was calculated when oocytes were switched from OR 7.4 to HCO₃⁻ 7.4 and then to 10% Na⁺ and HCO₃⁻, pH 6.4. Error bars represent means ± SE (n = 7).

Fig. 5.

SLC4A11 does not transport bicarbonate. HEK293 cells, grown on glass coverslips and transfected with cDNA encoding SLC4A11 or empty vector, were incubated with the pH-sensitive dye BCECF-AM. Coverslips were placed in a fluorescence cuvette and fluorescence was monitored in a fluorimeter, using λ_excitation = 440 and 502.5 nm and λ_emission = 528.7 nm. A: cuvette was perfused alternately with Cl⁻-containing (shaded bar) and Cl⁻-free (open bar) Ringer's buffers, bubbled with 5% CO₂. Black and gray traces represent results from cells transfected with SLC4A11 (n = 9) and vector (pcDNA3.1, n = 7), respectively. B: mean of Cl⁻/HCO₃⁻ exchange activity (J_H⁺) calculated from the initial rate of pH_i change during perfusion with Cl⁻-free buffer. C: sodium-dependent bicarbonate transport activity. BCECF-loaded HEK293 cells, transiently expressing SLC4A11 (black trace, n = 16) or empty vector (gray trace n = 12), were equilibrated in Na⁺-free Ringers buffer (gray bar), containing 5 μM ethyl-isopropyl amiloride (EIPA). All buffers in this experiment were bubbled with 5% CO₂. NH₄Cl was added (black bar) and then perfusion began with NH4Cl and Na⁺-free Ringers buffer (gray bar). Finally, cuvette was perfused with Na⁺-containing Ringer's buffer (white bar). D: Na⁺-dependent bicarbonate transport activity estimated by [H⁺] flux (J_H⁺), following switching to Na⁺-containing medium (Student's t-test; all comparisons not significant for SLC4A11 vs. control at each tested pH value).

Fig. 6.

SLC4A11 mediates extracellular pH-dependent NH₃ permeability in oocytes. SLC4A11 cRNA-injected or native oocytes were incubated for 72 h. pH and current were simultaneously measured with a double-barreled microelectrode combined with a two-electrode voltage clamp (clamped to −40 mV). A: concentration of protons [H⁺]_i was measured as the SLC4A11 (top) or native (bottom) expressing oocytes were exposed to oocyte Ringer's solution (OR) at pH 7.4, followed by OR containing 10 mM NH₄Cl pH 7.4 (black bar), OR pH 6.4 followed by OR containing 10 mM NH₄Cl pH 6.4, and OR pH 8.4 followed by OR containing 10 mM NH₄Cl pH 8.4. Experiments were performed in the nominal absence of CO₂/HCO₃⁻. B: current was measured when the native or SLC4A11-expressing oocytes were exposed to oocyte Ringer's solution (OR) at pH 7.4 followed by OR containing 10 mM NH₄Cl pH 7.4 (black line), OR pH 6.4 followed by OR containing 10 mM NH₄Cl pH 6.4, and OR pH 8.4 followed by OR containing 10 mM NH₄Cl pH 8.4. C: rate of [H⁺] change was calculated when oocytes were switched from OR to 10 mM NH₄Cl at pH 7.4, 6.4, and 8.4. D: membrane current movement mediated by SLC4A11 was calculated when oocytes were switched from OR to OR containing 10 mM NH₄Cl at pH 7.4, 6.4, and 8.4. Error bars represent means ± SE (n = 5–7). ***Significant difference (P ≤ 0.0001) compared with SLC4A11-expressing oocytes at same pH.

Fig. 7.

I–V relationships of native and SLC4A11-expressing oocytes in the presence of NH₄Cl. In native oocytes and SLC4A11-expressing oocytes, the current was recorded with a holding potential (V_h) of −40 mV, indicating the membrane conductance while perfused with Ringer's solution (control) and Ringer's solution containing 10 mM NH₄Cl at pH 7.4, 6.4, and 8.4. Experiments were performed in the nominal absence of CO₂/HCO₃⁻. Current-voltage relationships of NH₄Cl-induced membrane currents, as obtained from experiments in Fig. 6, C and D, is shown for both for both native (A) and SLC4A11-expressing (B) oocytes. Error bars represent means ± SE (n = 5–7).

Fig. 8.

Net current-voltage (I–V) relationships in the presence of NH₄Cl. Native oocytes and SLC4A11-expressing oocytes were voltage clamped to the indicated membrane potentials (V_m), and current was monitored during perfusion with oocyte Ringer's buffer (control), or oocyte Ringer's buffer, containing 10 mM NH₄Cl at pH 7.4 (NH₄Cl) (A) or pH 8.4 (B). Experiments were performed in the nominal absence of CO₂/HCO₃⁻. Difference in current between SLC4A11-expressing and native oocytes was calculated in the absence (control) or presence (NH₄Cl) of 10 mM NH₄Cl. Reversal potentials (inset, E_rev) were calculated from the x-intercept values.

Fig. 9.

Ammonia transport facilitated by SLC4A11. HEK293 cells, grown on glass coverslips and transfected with cDNA encoding SLC4A11 (n = 16) or empty vector (pcDNA3.1, n = 12), were incubated with the pH-sensitive dye BCECF-AM. Coverslips were placed in a fluorescence cuvette, and fluorescence was monitored in a fluorimeter by using λ_excitation = 440 and 502.5 nm and λ_emission = 528.7 nm. Cuvette was perfused with Na⁺-free Ringer's buffer, containing 5 μM ethyl-isopropyl amiloride (EIPA) (white bar), followed by the same buffer, containing 40 mM NH₄Cl (black bar). A: representative traces of intracellular pH (pH_i) changes. B: intracellular steady-state pH corresponding to the initial equilibrium in Ringer's buffer (pH_ini), alkalinization following NH₄Cl perfusion (pH_alk), and acidification following NH₄Cl withdrawal (pH_acid) states for pcDNA3.1-transfected (gray circles) and SLC4A11-expressing (black circles) cells. C: average [H⁺] flux (J_H+) during alkalinization and acidification were calculated during the initial perfusion with NH₄Cl-containing or following switch to NH₄Cl-free buffer, respectively. Both groups were compared at the same pH for alkalinization (pH = 7.30) and acidification (pH = 7.50) (Student's t-test, *P < 0.05 for SLC4A11 vs. control).