Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO 3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I NBC ) by two-electrode voltage clamp (Day 3), injected CA II protein ؉ Tris or just Tris (Day 3), measured I NBC or the initial rate at which the intracellular pH fell (dpH i /dt) upon applying 5% CO 2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I NBC or dpH i /dt (Day 4). Because dpH i /dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I NBC slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5 end of NBCe1-A and CA II at the 3 end (EGFP-e1-CAII). We measured I NBC or dpH i /dt (days 3-4), exposed oocytes to EZA, and measured I NBC or dpH i /dt (Day 3-4). dpH i /dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.The electrogenic Na/bicarbonate cotransporter (NBCe1 or e1) 3 plays a central role in HCO 3 Ϫ reabsorption and regulation of intracellular pH (1). The kidney-specific splice variant NBCe1-A is localized at the basolateral membrane of renal proximal tubule cells (2), where it mediates efflux of HCO 3 Ϫ (and/or CO 3 ϭ ). Cytoplasmic HCO 3 Ϫ arises from the intracellular hydration of CO 2 , which is catalyzed by the cytoplasmic enzyme carbonic anhydrase II (CA II) (3).Because the reports of Vince and Reithmeier (4 -6) that cytosolic CA II binds to the LDADD motif on the cytoplasmic C terminus of the Cl-HCO 3 exchanger AE1, Sterling et al. (7,8) have measured rates of intracellular pH (pH i ) change in HEK293 cells transiently transfected with AE1 and concluded that CA II enhances AE1-mediated HCO 3 Ϫ transport. The C termini of all three NBCe1 splice variants, i.e. NBCe1-A as well as the more universally expressed variant NBCe1-B (9, 10) and the brain-specific NBCe1-C (11), have two motifs similar to LDADD in AE1. Moreover, isothermal titration calorimetry and pulldown assays suggest that, at least under non-reducing conditions, the common C terminus of NBCe1-A/B interacts with CA II in vitro (12). Similarly, Pushkin and co-workers (12-15) working with a mouse proximal convoluted tubule (mPCT) cell line stably transfected with NBCe1-A, concluded that CA II enhances the current carried by NBCe1-A.For three reasons, we set out to verify the hypothesis that CA II enhances the activity of NBCe1-A. First, a pH i measurement, as in the AE1 study, is an indirect index of the rate of HCO 3 Ϫ ...

The human electrogenic renal Na-HCO(3) cotransporter (NBCe1-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 HCO(3)(-) from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-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 NBCe1-A (WT) and mutant NBCe1-A (R881C), 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 NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

The SLC4A10 gene product, commonly known as NCBE, is highly expressed in rodent brain and has been characterized by others as a Na ؉ -driven Cl-HCO 3 exchanger. However, some of the earlier data are not consistent with Na ؉ -driven Cl-HCO 3 exchange activity. In the present study, northern blot analysis showed that, also in humans, NCBE transcripts are predominantly expressed in brain. In some human NCBE transcripts, splice cassettes A and/or B, originally reported in rats and mice, are spliced out. In brain cDNA, we found evidence of a unique partial splice of cassette B that is predicted to produce an NCBE protein with a novel C terminus containing a protein kinase C phosphorylation site. We used pH-sensitive microelectrodes to study the molecular physiology of human NCBE expressed in Xenopus oocytes. In agreement with others we found that NCBE mediates the 4,4-diisothiocyanato-stilbene-2,2-disulfonic acid-sensitive, Na ؉ -dependent transport of HCO 3 ؊ . For the first time, we demonstrated that this transport process is electroneutral. Using Cl ؊ -sensitive microelectrodes positioned at the oocyte surface, we found that, unlike both human and squid Na ؉ -driven Cl-HCO 3 exchangers, human NCBE does not normally couple the net influx of HCO 3 ؊ to a net efflux of Cl ؊ .Moreover we found that that the 36 Cl efflux from NCBE-expressing oocytes, interpreted by others to be coupled to the influx of Na ؉ and HCO 3 ؊ , actually represents a CO 2 /HCO 3 ؊ -stimulated Cl ؊ self-exchange not coupled to either Na ؉ or net HCO 3 ؊ transport. We propose to rename NCBE as the second electroneutral Na/HCO 3 cotransporter, NBCn2.

The reported sequences of the human and mouse Na+-driven Cl-/HCO3(-) exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5'- and 3'-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D'. The novel NDCBE-C/D/D' transcripts initiate at a novel "exon 0" positioned approximately 35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D' protein products are predicted to be truncated by 54 aa in the cytosolic NH(2) terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25-50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.

Mutations in the electrogenic Na+/nHCO3- cotransporter (NBCe1, SLC4A4) cause severe proximal renal tubular acidosis, glaucoma, and cataracts in humans, indicating NBCe1 has a critical role in acid-base homeostasis and ocular fluid transport. To better understand the homeostatic roles and protein ontogeny of NBCe1, we have cloned, localized, and downregulated NBCe1 expression in zebrafish, and examined its transport characteristics when expressed in Xenopus oocytes. Zebrafish NBCe1 (zNBCe1) is 80% identical to published mammalian NBCe1 cDNAs. Like other fish NBCe1 clones, zebrafish NBCe1 is most similar to the pancreatic form of mammalian NBC (Slc4a4-B) but appears to be the dominant isoform found in zebrafish. In situ hybridization of embryos demonstrated mRNA expression in kidney pronephros and eye by 24 h postfertilization (hpf) and gill and brain by 120 hpf. Immunohistochemical labeling demonstrated expression in adult zebrafish eye and gill. Morpholino knockdown studies demonstrated roles in eye and brain development and caused edema, indicating altered fluid and electrolyte balance. With the use of microelectrodes to measure membrane potential (Vm), voltage clamp (VC), intracellular pH (pH(i)), or intracellular Na+ activity (aNa(i)), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCe1s, demonstrating electrogenic Na+ and HCO3- transport as well as similar drug sensitivity, including inhibition by 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and tenidap. These data indicate that NBCe1 in zebrafish shares many characteristics with mammalian NBCe1, including tissue distribution, importance in systemic water and electrolyte balance, and electrogenic transport of Na+ and HCO3-. Thus zebrafish promise to be useful model system for studies of NBCe1 physiology.