Characterization of pH i Regulation by NBCe1/SLC4A4 Variants with Known Clinical Phenotypes in Human Immortalized Trabecular Meshwork Cells with a Red‐Shifted Genetically‐Encoded pH‐Indicator “pHire”

The electrogenic Na + /HCO 3 − cotransporter (NBCe1/SLC4A4) maintains systemic acid/base balance by reabsorbing HCO 3 − in the renal proximal tubule but also contributes to intracellular pH (pH i ) regulation in the eye, nervous system, pancreas, and intestine. Human recessive mutations lead to diverse phenotypes (glaucoma, proximal renal tubular acidosis, poor dentition) and mouse models suggest some organ manifestations are partially penetrant. The objective of this study was to develop a full spectrum genetically‐encoded live‐imaging toolkit which could be adapted to assay NBCe1‐mediated pH i regulation across a variety of cell types to further characterize how NBCe1 recessive mutations and polymorphisms functionally interact with each other and wild‐type (WT) NBCe1 to produce alterations in cellular acid/base physiology. WT human NBCe1 (WT‐NBCe1) and a recently characterized human recessive mutation (Mut‐NBCe1) were tagged with GFP. Each construct was co‐expressed via transfection in human immortalized trabecular meshwork cells (TM5) with “pHire”, a red‐shifted (556/630nm ex/em) pH‐sensitive variant of mApple, to assay NBCe1 function through real‐time quantification of pH i via widefield epifluorescent live‐imaging. pHire successfully reported pH i of TM5 cells in response to H + loading with NH 4 Cl pulses. Calibration of pHire revealed an in vitro p K a of 7.44, a 5‐fold linear fluorescence response from pH 6.5 to 8.0, and stability following acid loads (pH 5.0) and long imaging protocols (30 min). pH i of TM5 cells fell in response to CO 2 /HCO 3 − — buffered solution and displayed a modest Na + ‐dependent recovery ( dpH i / dt = 0.02 ± 0.01 pH units min −1 in CO 2 /HCO 3 − , −0.06 ± 0.02 in Na + ‐free), consistent with intrinsic TM5‐cell NBCe1 activity. Transfection of WT‐NBCe1 did not consistently alter HCO 3 − recovery but accelerated pH i decrease in Na + ‐free solution by 10‐fold (1017 ± 3%) relative to neighboring untransfected cells. Transfection with E91R‐NBCe1 failed to similarly potentiate acidification upon Na + withdrawal (117 ± 81%), and transfection with Mut‐NBCe1 suppressed Na + ‐dependent acidification to 72 ± 28% of untransfected cells. These data suggest that although both Mut‐NBCe1 and E91R‐NBCe1 are functionally “transporter‐null” they possess different capacities to impair endogenous WT‐NBCe1, possibly through dimerization and localization deficits. Such differences could manifest discretely in specific cell types and may underlie the partial penetrance of some NBCe1‐associated phenotypes. Support or Funding Information Funding: T32‐DK007013 to AJR, DK092408, DK100227 and R25‐DK101405 to MFR.