SLC4A11 is an EIPA-sensitive Na(+) permeable pHi regulator

Abstract

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.

Keywords: Na+ permeability; SLC4A11; bicarbonate; borate; pH.

Fig. 1.

SLC4A11 expression and localization. A: expression of the recombinant protein in HEK 293 cells transfected with phCMV2-HA-SLC4A11 at 24, 48, and 72 h. Detection was performed with anti-SLC4A11 antibody. SLC4A11 is detected as a 120-kDa band. B: average band density (n = 3). C: sulfo-NHS-S-S-Biotin (SNSB) Membrane preparation of SLC4A11-transfected cells. Detection was performed with anti-HA antibody. Total protein fraction (T), unbound protein fraction (U), and bound eluted membrane protein fraction (B). The presence of the protein in the eluted fraction indicates that the protein is localized in the membrane. D: immunohistochemistry using anti-HA in SLC4A11-transfected HEK293 shows membrane localization of the recombinant protein. E: immunohistochemistry using anti-HA in empty vector-transfected [control (Ctrl)] HEK293 cells. F: RT-PCR for pNBC and kNBC bicarbonate transporters in HEK293 cells and positive controls. G: RT-PCR for NHE1–5 in HEK293 cells.

Fig. 2.

Effect of bicarbonate on pH_i in SLC4A11-transfected HEK293. A: cells were loaded with BCECF-AM and pH_i changes were analyzed in bicarbonate-free (BF) and bicarbonate-rich Ringer (BR). Traces represent the average of 12 experiments. Inset: pH_i changes in bovine corneal endothelial cells (BCE). B: steady-state pHi in BF and BR conditions. C: delta pH_i due to CO₂-induced acidification (points 2–3 in A). D: initial maximum rate of alkalinization recovery was obtained by linear regression on the first 30 s of change. †P < 0.05, significantly different to paired BF condition. Labels 1–4 are explained on the text.

Fig. 3.

Effect of bicarbonate on intracellular Na⁺ concentration ([Na⁺]_i) in SLC4A11-transfected HEK293. A: [Na⁺]_i in response to BR addition. Traces represent the average of 6 experiments. B: average [Na⁺]_i at the steady state in BF and BR conditions. *P < 0.05, significantly different from control.

Fig. 4.

Effect of sodium-free BR perfusion on pHi in SLC4A11-transfected HEK293. A: pHi responses to Na free perfusion in BR for SLC4A11 or empty vector (control)-transfected HEK293 cells. Traces represent the average of 6 experiments. B: summary of rates of acidification (Na⁺-free). C: extent of acidification. D: rate of alkalinization (Na⁺ add back). E: extent of alkalinization.

Fig. 5.

Recovery from acid load in SLC4A11-transfected cells. A: alkalinization, acidification, and recovery from acid load in SLC4A11 or empty vector (control)-transfected HEK293 cells following exposure (2 min) and removal of 10 mM NH₄Cl in BF Ringer. Traces represent the average of 7 experiments. B: NH₄⁺-induced acidification was calculated by linear regression of the first 20 s of pH change. C: delta pHi following NH₄⁺ removal. D: rate of alkalinization recovery was calculated by linear regression on the first 30 s of pH change corresponding to pH: 7.25 in BF. Rate of alkalinization recovery in presence of 5-(N-ethyl-N-isopropyl) amiloride (EIPA; 1 μM) was calculated by linear regression at matched pH: 7.25 and does not represent in this case the initial rate. *P < 0.05, significantly different from control. †P < 0.05, significantly different in the presence of EIPA. Labels 1–10 are explained in the text.

Fig. 6.

EIPA dose-response analysis of SLC4A11-transfected HEK293 cells. Recovery from NH₄⁺ pulse was measured in SLC4A11 and empty vector (control)-transfected HEK293 cells in the presence of 0, 0.1, 0.5, 1, and 10 μM EIPA. Each point represents the average of 6 experiments. Data were fitted to a 4-parameter EC₅₀ function.

Fig. 7.

Effect of Na⁺-free BF perfusion on pHi in SLC4A11-transfected and control HEK293. A: pH_i decrease in response to 5 min of Na⁺-free Ringer. Traces represent the average of 6 experiments. B: acidification rate on Na⁺ removal. C: extent of acidification. D: alkalinization rate on Na⁺ add back. E: extent of pH_i change on Na⁺ add back. *P < 0.05. F: effect of EIPA (1 μM) on pHi recovery following Na⁺-free acidification in SLC4A11 or empty vector (control) HEK293-transfected cells. Traces represent the average of 6 experiments.

Fig. 8.

Sodium fluxes following a 5-min sodium free pulse in BF in SLC4A11-transfected and control HEK293. A: [Na⁺]_i in response to Na⁺ removal and add back in BF. Traces represent the average of 8 experiments. B: resting [Na⁺]_i in bicarbonate-free Ringer. C: Na⁺ efflux rates. D: Na⁺ influx rates. *P < 0.05.

Fig. 9.

Ammonium pulse and acidification in control and SLC4A11-transfected PS120 cells. A: pH_i responses during and following brief perfusion with 10 mM NH₄Cl. Traces represent the average of 6 experiments. B: average ± SE of rate of acidification in presence of NH₄⁺. *P < 0.05.

Fig. 10.

Recovery from acid load in control and SLC4A11-transfected PS120 cells. The duration of the ammonium pulse was adjusted to provide more similar acidification in control and-transfected PS120 cells. A: alkalinization, acidification, and recovery from acid load in SLC4A11 or empty vector (control)-transfected PS120 cells following exposure and removal of NH₄Cl in BF Ringer in presence or absence of EIPA 10 μM. SLC4A11-transfected cells were acidified by incubation for 2 min with 10 mM NH₄Cl. To acidify control cells to the same extent, 4 min incubation with 20 mM NH₄Cl was required. Traces represent the average of 8 experiments. B: average ± SE of ΔpH_i of acidification after NH₄ removal. C: rate of rate of alkalinization recovery. *P < 0.05.

Fig. 11.

EIPA dose-response analysis of SLC4A11-transfected PS120 cells. Recovery from NH₄⁺ pulse was measured in SLC4A11-transfected PS120 cells in the presence of 0, 0.01, 0.1, 0.5, 1, 10, and 20 μM EIPA. Each point represents the average of 6 experiments. Data were fitted to a 4-parameter EC₅₀ function.

Fig. 12.

NMDG-based Na⁺-free Ringer to test Borate transport. Absence of borate dependent proton flux in SLC4A11-transfected and control HEK293 cells. A: pH_i responses to Na⁺-free pulse in the presence or absence of 10 mM borate. Traces represent the average of 6 experiments. B: average ± SE of rate of acidification with and without borate.

Fig. 13.

TMA-based Na⁺-free Ringer to test borate transport. Absence of borate dependent proton flux in SLC4A11-transfected and control HEK293 cells. A: pH_i responses to Na⁺-free pulse in the presence or absence of 10 mM borate. Traces represent the average of 7 experiments. B: average ± SE of rate of acidification with and without borate. C: extent of acidification. D: rate of alkalinization on Na⁺ add back. E: extent of alkalinization on Na⁺ add back.

Fig. 14.

Sequence analysis of SLC4A11. A: phylogenetic tree showing relationship between SLC4, NHE, and Amiloride-sensitive Na⁺ transporter families of ion transporters. Sequences were aligned with ClustalW2 program and the alignment was loaded in Jalview 2.7 software (http://jalview.org) to generate the phylogenetic tree showing evolutionary relationship between the proteins. Numbers at the nodes and the lengths of the lines indicate relative evolutionary distance. B: SLC4 family alignment of the first hinge loop (top) and the second hinge loop (bottom). This 1st hinge loop is located in the EL5 of SLC4A11 between TM9 and TM10. The 1st hinge loop is postulated to have sodium attracting amino acids. E737 (Glutamine, net negative charge) is conserved in all members of the SLC4 family that transport sodium and also in SLC4A11. The 2nd hinge loop is located in the TM12 domain of SLC4A11. This region it is postulated to have anion attracting motifs. R804, R822, and K828 (arginine and lysine, net positive charge) are conserved in SLC4A11.