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SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH 3 sensitive, electrogenic H + transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH 4 Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH 4 Cl when compared to wild type, indicating a decreased H + permeability in mutants. All mutants exhibited significantly reduced H + currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH 4 Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H + flux properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na(+), H(+) (OH(-)), bicarbonate, borate, and NH4 (+). Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH4 (+), Na(+), and H(+) contributions to electrogenic ion transport through SLC4A11 stably expressed in Na(+)/H(+) exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH4Cl were determined by the [NH3]o, not [NH4 (+)]o, and current amplitudes varied with the [H(+)] gradient. These currents were relatively unaffected by removal of Na(+), K(+), or Cl(-) from the bath but could be reduced by inclusion of NH4Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH4Cl. Reversal potential shifts in response to changing [NH3]o and pHo suggested an NH3/H(+)-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH3 but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH3/H(+)). NH3-dependent currents were insensitive to 10 μM ethyl-isopropyl amiloride or 100 μM 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid. We propose that SLC4A11 is an NH3/2H(+) co-transporter exhibiting unique characteristics., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter. CO2/HCO3(-) perfusion yielded no significant differences in rate or extent of pHi changes or Na(+) flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3(-), acidification on removal of Na(+) and alkalinization on Na(+) add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na(+)-HCO3(-) cotransport activity. In the absence of CO2/HCO3(-), SLC4A11-transfected cells showed higher resting intracelllular Na(+) concentration ([Na(+)]i; 25 vs. 17 mM), increased NH4(+)-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na(+) efflux and influx were faster (80%) following Na(+) removal and add back, respectively, indicative of Na(+)-OH(-)(H(+)) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na(+)-OH(-)(H(+)) transporter and not an activator of NHEs. SLC4A11-mediated H(+) efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 μM). The presence of 10 mM borate did not alter dpHi/dt or ΔpH during a Na(+)-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na(+)-OH(-)(H(+)) and NH4(+) permeability.

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⁻.

Purpose: To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells., Methods: To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene expression following SLC4A11 silencing were determined using the RT(2)Profiler PCR array for human apoptosis while activation of the apoptotic pathway was ascertained by western analysis., Results: SLC4A11 silencing in HCECs could be achieved by stable expression of shRNA targeted against SLC4A11. SLC4A11 knockdown suppressed HCEC growth and reduced HCEC viability compared to the control. This reduction in cell growth is associated with increased apoptosis in SLC4A11-silenced cells., Conclusions: Our data suggest that the reduction of cell number with time in SLC4A11-depleted HCECs is due to an increase in cell death by apoptosis. This suggests that SLC4A11 is necessary for cell survival and may explain the pathologic corneal endothelial cell loss in endotheliopathies due to SLC4A11 mutations.

Purpose: Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium., Methods: RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing., Results: RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium., Conclusions: CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.

Purpose: To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport., Methods: BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2)., Results: Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response., Conclusions: Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.