Investigation of a Juxtamembrane Lysine‐rich Region as a Determinant of Intracellular pH Sensitivity in the H + ‐conducting Transmembrane Protein Slc4a11

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 (pH i ) and extracellular pH (pH e ). By aligning the amino‐acid sequences of mammalian Slc4a11 and their bicarbonate‐transporting paralogs, we have identified a unique and highly‐conserved insertion ‘ K x K xVG K ’ 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 pH i sensitivity of ROMK currents (Fakler et al. EMBO J, 1996). Thus, we hypothesized that K343 might contribute to the pH i 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 ( G m ) at pH e =7.5 by using two‐electrode voltage‐clamp circuitry while raising oocyte pH i (monitored by a H + ‐selective microelectrode) using a micro‐injector loaded with 1.2M NaHCO 3 solution. As controls we used oocytes that had been injected with water rather than cRNA (‘H 2 O‐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 G m of R125H‐expressing cells is indistinguishable from that of H 2 O‐injected cells over the entire tested pH I range (7.2–8.1), whereas the G m of WT‐expressing cells is significantly greater than that of H 2 O‐injected cells over the entire range (n=4–12 data points). On the other hand, the G m of K343Q‐expressing cells is indistinguishable from H 2 O‐injected cells over the pH i range 7.2–7.5, greater than H 2 O‐injected cells but not as great as WT‐expressing cells over the pH i range 7.5–7.9, and ultimately indistinguishable from WT‐expressing cells over the pH i range 7.9–8.1 (n = 4–35 data points). Thus the pH i ‐responsiveness of K343Q is altered compared to WT. The H + conductance of Slc4a11 is much enhanced at pH e =8.5 such that the pH i vs. G m relationship becomes sigmoidal and can be fit to the Hill equation to determine an apparent pK and Hill constant. We assessed K343Q at pH e =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 pH i sensitivity of Slc4a11. Further work is underway to identify other determinants of pH i and pH e sensitivity. Support or Funding Information This work was supported by NIH R01‐EY028580 to MDP This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .