The Effect of SLC4A11 Mutations on Intracellular pH

Abstract

SLC4A11 is a ubiquitously expressed transporter showing Na+‐OH−, NH3‐H+ cotransport, and water permeability. SLC4A11 mutations have been associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED2) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). To address the role of known human SLC4A11 mutations on cellular physiology we measured intracellular pH regulation in PS120 fibroblasts that have been stably transfected with SLC4A11 mutant variants.Mutations were made in the Wild‐Type (WT) construct phCMV2‐hSLC4A11 by site‐directed mutagenesis. Mutants with a single amino acid change were obtained and referred to as R125H, C386R, V507I and N639A respectively. PS120 cells were stably transfected with those constructs and empty vector (EV) using SuperFect, and selected by Geneticin. For intracellular pH (pHi) measurement, PS120 cells grown on coverslips were loaded with a pH‐sensitive fluorescent dye, 2′7′‐Bis‐(2‐Carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF‐AM). The coverslip was mounted in a perfusion chamber (37°C) on the stage of a fluorescence microscope. Perfusion was performed with a Bicarbonate‐free solution (BF) to obtain stable baseline pH, followed by pulsing cells with 10 mM NH4Cl for 5 minutes, and changed back to BF perfusion until pHi was stable. Fluorescence ratio (495/440 nm) was converted to pHi following calibration with high K+/Nigericin. Results are means ± SE from 3 trials.The baseline pHi was 7.211 ± 0.136, 7.877 ± 0.056, 7.391 ± 0.069, 7.226 ± 0.232, 7.504 ± 0.104 and 7.359 ± 0.221 for WT, EV, R125H, C386R, V507I and N639A, respectively. The pHi increase from NH4Cl pulse was 0.486 ± 0.014, 0.161 ± 0.018, 0.298 ± 0.05, 0.307 ± 0.07, 0.304 ± 0.011 and 0.442 ± 0.084 respectively. The rate of pHi increase (ΔpH/min) was 2.184 ± 0.33, 0.469 ± 0.037, 1.004 ± 0.124, 1.024 ± 0.362, 1.17 ± 0.142 and 1.818 ± 0.405, respectively. The subsequent rate of pHi decrease in the presence of NH4Cl was (ΔpH/min) 0.153 ± 0.038, 0.006 ± 0.012, 0.012 ± 0.005, 0.011 ± 0.003, 0.033 ± 0.002 and 0.009 ± 0.007, respectively. Removal of NH4Cl‐BF resulted in maximum acidification of 1.205 ± 0.122, 0.343 ± 0.074, 0.671 ± 0.019, 0.409 ± 0.122, 0.747 ± 0.041 and 0.748 ± 0.048 pH units. NH4Cl induced pHi changes were WT > N639A > V507I > R125H > C386R > EV.Expression of SLC4A11 WT and mutants lower resting pHi relative to EV, consistent with a H+ permeability. NH4Cl induced pHi changes were consistent with reduction of NH3:H+ linked cotransport in SLC4A11 mutants.Support or Funding InformationNIH R01 EY08834‐23 & P30EY019008