Investigation of a Juxtamembrane Lysine‐rich Region as a Determinant of Intracellular pH Sensitivity in the H+‐conducting Transmembrane Protein Slc4a11
Authors: Quade Bianca Natalia , Marshall Aniko , Parker Mark D 
Published: June 2021

SLC4A11 is a unique H+‐transporting member of the SLC4 family of bicarbonate transporters that is expressed in a variety of cells, including corneal endothelia. The H+ conductance of SLC4A11 increases in response to rises in both intracellular pH (pHi) and extracellular pH (pHe). By aligning the amino‐acid sequences of mammalian Slc4a11 and their bicarbonate‐transporting paralogs, we have identified a unique and highly‐conserved insertion ‘KxKxVGK’ in Slc4a11 at the juxtamembrane boundary between its cytosolic amino‐terminal‐domain and its first transmembrane‐span. The third lysine in this motif (‘K343’ in mouse Slc4a11) brings to mind the lysine at a comparable location in the renal K+‐channel ROMK that underlies the pHi sensitivity of ROMK currents (Fakler et al. EMBO J, 1996). Thus, we hypothesized that K343 might contribute to the pHi sensitivity of Slc4a11.To probe the role of K343, we generated a mutant ‘K343Q’, Q being the non‐titratable residue glutamine that is found at this location in other SLC4s. We expressed wild‐type (WT) or K343Q Slc4a11 from cRNA injected into Xenopus oocytes. We then compared the pH‐sensitivities of the expressed H+ conductances (Gm) at pHe=7.5 by using two‐electrode voltage‐clamp circuitry while raising oocyte pHi (monitored by a H+‐selective microelectrode) using a micro‐injector loaded with 1.2M NaHCO3 solution. As controls we used oocytes that had been injected with water rather than cRNA (‘H2O‐injected’) and oocytes that had been injected with cRNA encoding the mouse equivalent of the human corneal‐dystrophy‐causing SLC4A11 mutant ‘R125H’.ANOVA shows that the Gm of R125H‐expressing cells is indistinguishable from that of H2O‐injected cells over the entire tested pHI range (7.2–8.1), whereas the Gm of WT‐expressing cells is significantly greater than that of H2O‐injected cells over the entire range (n=4–12 data points). On the other hand, the Gm of K343Q‐expressing cells is indistinguishable from H2O‐injected cells over the pHi range 7.2–7.5, greater than H2O‐injected cells but not as great as WT‐expressing cells over the pHi range 7.5–7.9, and ultimately indistinguishable from WT‐expressing cells over the pHi range 7.9–8.1 (n = 4–35 data points). Thus the pHi‐responsiveness of K343Q is altered compared to WT.The H+ conductance of Slc4a11 is much enhanced at pHe=8.5 such that the pHi vs. Gm relationship becomes sigmoidal and can be fit to the Hill equation to determine an apparent pK and Hill constant. We assessed K343Q at pHe=8.5 and calculated a pK of 7.15 ± 0.02 and a Hill constant of 5 ± 1 (n = 4). Both of these values are significantly different from the WT values that we had previously determined (pK= 7.25 ± 0.03, Hill = 8 ± 1, n = 10). The lowered Hill and pK for K343Q are consistent with the loss of a titratable residue that contributes to the pHi sensitivity of Slc4a11. Further work is underway to identify other determinants of pHi and pHe sensitivity.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.