SLC4A11 is an EIPA-sensitive Na⁺ permeable pH_i 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/d t 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.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

“…Transfection and Cell Culture-Human SLC4A11-HAtagged or empty vector (EV) were stably transfected into NHEdeficient PS120 Chinese hamster CCL39 fibroblast cell line as described previously (9). The SLC4A11 (NP_001167561.1) cDNA was subcloned into a plasmid containing a neo r gene for Geneticin G418 selection and a C-terminal HA tag (13).…”

“…Intracellular pH Measurements-Intracellular pH measurements were performed as described previously (9). Briefly, PS120 cells were cultured on poly-L-lysine-coated 25- .…”

“…A more recent report has suggested H ϩ permeability through SLC4A11 but independent of Na ϩ (12). Interestingly, ammonium permeability (9) and water permeability (13) have also been suggested for SLC4A11, and the across-species highly conserved asparagine-proline-X (NPX) water channel motif has been identified in a transmembrane domain of SLC4A11 (N 639 PS) (13). Because several water channels, such as AQP3, AQP7, AQP8, AQP9, and AQP10, are ammonia-permeable (14), and a single-point mutation in the aromatic/arginine region (ar/R) can induce ammonia permeability in the ammonia-impermeable water channel AQP1 (15), we hypothesized that SLC4A11 may also exhibit ammonia permeability similar to AQPs.…”

“…Although SLC4A11 is classified as a member of the SLC4 (solute-linked co-transporter 4) family of bicarbonate transporters, it shares rather low (14 -20%) homology with other family members. Phylogenetic analysis of SLC4A11 indicates a separate branch of early evolutionary divergence in the SLC4 family, suggesting a divergent function (9).…”

“…Initial studies suggested that SLC4A11 is an electrogenic 2Na ϩ -OH Ϫ (or borate) co-transporter (10). However, more recent studies provided evidence that human SLC4A11 transports neither borate nor bicarbonate (9,11). These studies suggested ethyl-isopropylamiloride (EIPA) 3 -sensitive Na ϩ and H ϩ (or OH Ϫ ) permeability through SLC4A11; however, a precise stoichiometry could not be determined.…”