Investigating the Intracellular and Extracellular pH Sensitivities of the H+‐conducting Transmembrane Protein Slc4a11

Abstract

SLC4A11 is the only member of the SLC4 family that transports H+ rather than bicarbonate (HCO3−) or carbonate (CO3=). SLC4A11 is expressed in a variety of cells including the corneal endothelial cells (CECs) in which it is presumed to mediate H+ influx. CECs help to maintain the optimal hydration status and optical clarity of the cornea by pumping fluid from the corneal stroma. This pump function requires the joint action of NBCe1 (Na+/CO3= influx), MCT1 (H+/lactate− influx), and SLC4A11. Notably, SLC4A11 mutations are sufficient to cause pump failure and the subsequent corneal swelling and opacification that are characteristic of corneal endothelial dystrophy. The role of SLC4A11 in corneal health and disease remains unclear, although we have hypothesized that SLC4A11 action could promote CEC health by correcting pH disturbances caused by imbalanced NBCe1 (base) and MCT1 (acid) import. Consistent with this hypothesis, our laboratory has previously demonstrated that the magnitude of the H+ conductance mediated by SLC4A11 is increased by rises in intracellular pH (pHi) as well as rises in extracellular pH (pHe).To better understand the features of SLC4A11 activation, we studied the pH‐sensitivity of mouse Slc4a11 expressed from cRNA in Xenopus oocytes. Using two electrode voltage clamp circuitry in conjunction with a H+‐selective microelectrode and a micro‐injector loaded with 1.2M NaHCO3 solution, we caused incremental increases in oocyte pHi and followed the accompanying increase in membrane conductance (Gm) while pHe was maintained at 7.5 or 8.5. We find, when pHe = 7.5, that Gm rises steadily over the pHi range 7.1–8.1, reaching a maximum observed conductance (Gm(max)) of 82 ± 5 μS (n = 6, SE) with an apparent intracellular pK ~7.6. On the other hand, when pHe= 8.5, Gm exhibits a steeper dependence on pHi and rises in a sigmoidal fashion over the pHi range 7.0–7.4 achieving a Gm(max) of 78 ± 8 μS (n = 14, SE) at pHi ≥ 7.4. [In comparable experiments performed on oocytes that were not expressing Slc4a11, Gm for was not greater than 4 μS (n=6) at pHi > 7.4.] The data gathered from Slc4a11‐expressing oocytes at pHe=8.5 can be fit to the Hill equation to reveal an average intracellular pK of 7.15 ± 0.02 (n = 10, SE) and an average Hill constant of 8 ± 1 (n = 10, SE).These data reveal a complex connection between pHe and pHi− sensitivity which highlights the challenge of working with such a dynamic substrate. Specifically, our data indicate that the kinetics of pHi sensing by Slc4a11 are modulated by pHe and are governed by numerous titratable residues. Knowledge of the kinetics governing the pH‐sensitivity of SLC4A11 H+ conductance will be valuable for assessing the physiological relevance of pH sensing by SLC4A11, for implementing SLC4A11 in mathematical models of CEC action, and for assessing the consequences of SLC4A11 mutations.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.