Corneal dystrophy mutations R125H and R804H disable SLC4A11 by altering the extracellular pH dependence of the intracellular pK that governs H+(OH-) transport.

Mutations in the H+(OH-) conductor SLC4A11 result in corneal endothelial dystrophy. In previous studies using mouse Slc4a11, we showed that the pK value that governs the intracellular pH dependence of SLC4A11 (pKi) is influenced by extracellular pH (pHe). We also showed that some mutations result in acidic or alkaline shifts in pKi, indicating that the pH dependence of SLC4A11 is important for physiological function. An R125H mutant, located in the cytosolic amino terminus of SLC4A11, apparently causes a complete loss of function, yet the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) can partially rescue SLC4A11/R125H activity. In the present study we set out to determine whether the effect of R125H is explained by an extreme shift in pKi. In Xenopus oocytes, we measured SLC4A11-mediated H+(OH-) conductance while monitoring pHi. We find that 1) the human corneal variant SLC4A11-B has a more acidic pKi than mouse Slc4a11, likely due to the presence of an NH2-terminal appendage; 2) pKi for human SLC4A11 is acid-shifted by raising pHe to 10.00; and 3) R125H and R804H mutants mediate substantial H+(OH-) conductances at pHe = 10.00, with pKi shifted into the wild-type range. These data suggest that the defect in each is a shift in pKi at physiological pHe, brought about by a disconnection in the mechanisms by which pHe influences pKi. Using de novo modeling, we show that R125 is located at the cytosolic dimer interface and suggest that this interface is critical for relaying the influence of pHe on the external face of the transmembrane domain to the intracellular, pKi-determining regions.

The molecular identity of the characean OH- transporter: a candidate related to the SLC4 family of animal pH regulators.

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH-, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH-/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH-/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3- Cotransporter NBCe1.

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+-2HCO3- stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3- influx. However, NBCe1 in the kidney mediates HCO3- efflux (HCO3- reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3- efflux) than in murine colons (in which NBCe1 mediates HCO3- influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

The therapeutic importance of acid-base balance.

Baking soda and vinegar have been used as home remedies for generations and today we are only a mouse-click away from claims that baking soda, lemon juice, and apple cider vinegar are miracles cures for everything from cancer to COVID-19. Despite these specious claims, the therapeutic value of controlling acid-base balance is indisputable and is the basis of Food and Drug Administration-approved treatments for constipation, epilepsy, metabolic acidosis, and peptic ulcers. In this narrative review, we present evidence in support of the current and potential therapeutic value of countering local and systemic acid-base imbalances, several of which do in fact involve the administration of baking soda (sodium bicarbonate). Furthermore, we discuss the side effects of pharmaceuticals on acid-base balance as well as the influence of acid-base status on the pharmacokinetic properties of drugs. Our review considers all major organ systems as well as information relevant to several clinical specialties such as anesthesiology, infectious disease, oncology, dentistry, and surgery.

pH dependence of the Slc4a11-mediated H+ conductance is influenced by intracellular lysine residues and modified by disease-linked mutations.

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.

Extrarenal Signs of Proximal Renal Tubular Acidosis Persist in Nonacidemic Nbce1b/c-Null Mice.

BACKGROUND: The SLC4A4 gene encodes electrogenic sodium bicarbonate cotransporter 1 (NBCe1). Inheritance of recessive mutations in SLC4A4 causes proximal renal tubular acidosis (pRTA), a disease characterized by metabolic acidosis, growth retardation, ocular abnormalities, and often dental abnormalities. Mouse models of pRTA exhibit acidemia, corneal edema, weak dental enamel, impacted colons, nutritional defects, and a general failure to thrive, rarely surviving beyond weaning. Alkali therapy remains the preferred treatment for pRTA, but it is unclear which nonrenal signs are secondary to acidemia and which are a direct consequence of NBCe1 loss from nonrenal sites (such as the eye and enamel organ) and therefore require separate therapy. SLC4A4 encodes three major NBCe1 variants: NBCe1-A, NBCe1-B, and NBCe1-C. NBCe1-A is expressed in proximal tubule epithelia; its dysfunction causes the plasma bicarbonate insufficiency that underlies acidemia. NBCe1-B and NBCe1-C exhibit a broad extra-proximal-tubular distribution. METHODS: To explore the consequences of Nbce1b/c loss in the absence of acidemia, we engineered a novel strain of Nbce1b/c-null mice and assessed them for signs of pRTA. RESULTS: Nbce1b/c-null mice have normal blood pH, but exhibit increased mortality, growth retardation, corneal edema, and tooth enamel defects. CONCLUSIONS: The correction of pRTA-related acidemia should not be considered a panacea for all signs of pRTA. The phenotype of Nbce1b/c-null mice highlights the physiologic importance of NBCe1 variants expressed beyond the proximal tubular epithelia and potential limitations of pH correction by alkali therapy in pRTA. It also suggests a novel genetic locus for corneal dystrophy and enamel hypomineralization without acidemia.