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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 4,40-diisothiocyano-2,20-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The SLC4 family Cl−/HCO3− cotransporters (NBCe1, NBCe2, NBCn1, and NBCn2) contribute to a variety of vital physiological processes including pH regulation and epithelial fluid secretion. Accordingly, their dysfunction can have devastating effects. Disorders such as epilepsy, hemolytic anemia, glaucoma, hearing loss, osteopetrosis, and renal tubular acidosis are all genetically linked to SLC4-family gene loci. This review summarizes how studies of Slc4-modified mice have enhanced our understanding of the etiology of SLC4-linked pathologies and the interpretation of genetic linkage studies. The review also surveys the novel disease signs exhibited by Slc4-modified mice which could either be considered to presage their description in humans, or to highlight interspecific differences. Finally, novel Slc4-modified mouse models are proposed, the study of which may further our understanding of the basis and treatment of SLC4-linked disorders of HCO3−-transporter dysfunction. [ABSTRACT FROM AUTHOR]

The SLC4A11 gene encodes the bicarbonate-transporter-related protein BTR1, which is mutated in syndromes characterized by vision and hearing loss. Signs of these diseases [congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome] are evident in mouse models of Slc4a11 disruption. However, the intrinsic activity of Slc4a11 remains controversial, complicating assignment of its (patho)physiological role. Most studies concur that Slc4a11 transports H+ (or the thermodynamically equivalent species OH-) rather than HCO3- , but disparities have arisen as to whether the transport is coupled to another species such as Na+ or NH3/NH4+ . Here for the first time, we examine the action of mouse Slc4a11 in Xenopus oocytes. We simultaneously monitor changes in intracellular pH, membrane potential, and conductance as we alter extracellular pH, revealing the electrical and chemical driving forces that underlie the observed ion fluxes. We find that mSlc4a11 is an ideally selective H+/OH- conductive pathway, the action of which is uncoupled from the cotransport of any other ion. We also find that the activity of mSlc4a11 is independently enhanced by both extracellular and intracellular alkalinization, suggesting OH- as the most likely substrate and providing a novel explanation for the apparent NH3-dependence of Slc4a11-mediated currents reported by others. We suggest that the unique properties of Slc4a11 action underlie its value as a pH regulator in corneal endothelial cells. [ABSTRACT FROM AUTHOR]

Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic aminoterminus (Nt) of NBCe1-C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCe1-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCe1 variant. In the present study, we performed two-electrode voltageclamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCe1-B construct that lacks amino acid residues 2-16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical. [ABSTRACT FROM AUTHOR]

The human electrogenic renal Na-HCO3 cotransporter (NBCeI-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO3- from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCeI-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na~ affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCeI-A (WT) and mutant NBCeI-A (R88IC), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCeI-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Measuring ion transport activities in Xenopus oocytes using the ion-trap technique," by Maxime Blanchard and colleagues in the 2008 issue.