G2Cdb::Gene report

Gene id
G00001367
Gene symbol
SLC4A4 (HGNC)
Species
Homo sapiens
Description
solute carrier family 4, sodium bicarbonate cotransporter, member 4
Orthologue
G00000118 (Mus musculus)

Databases (7)

Gene
ENSG00000080493 (Ensembl human gene)
8671 (Entrez Gene)
456 (G2Cdb plasticity & disease)
SLC4A4 (GeneCards)
Literature
603345 (OMIM)
Marker Symbol
HGNC:11030 (HGNC)
Protein Sequence
Q9Y6R1 (UniProt)

Synonyms (5)

  • HNBC1
  • NBC1
  • NBC2
  • hhNMC
  • pNBC

Literature (46)

Pubmed - other

  • Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results.

    Dorfman R, Li W, Sun L, Lin F, Wang Y, Sandford A, Paré PD, McKay K, Kayserova H, Piskackova T, Macek M, Czerska K, Sands D, Tiddens H, Margarit S, Repetto G, Sontag MK, Accurso FJ, Blackman S, Cutting GR, Tsui LC, Corey M, Durie P, Zielenski J and Strug LJ

    Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON, Canada. ruslan.dorfman@gmail.com

    Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16-20% of CF newborns, providing linkage and association results from large family and case-control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case-control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy-Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.

    Funded by: NHGRI NIH HHS: HG-0004314, R03 HG004314, R03 HG004314-02; NHLBI NIH HHS: R01 HL068927, R01 HL068927-08

    Human genetics 2009;126;6;763-78

  • Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport.

    Yang HS, Kim E, Lee S, Park HJ, Cooper DS, Rajbhandari I and Choi I

    Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO(3) transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO(3) transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO(3)(-) currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO(3)-dependent pH recovery from a CO(2)-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO(2)/HCO(3)(-)-free solution that was abolished by Cl(-) removal from the bath. In the presence of CO(2)/HCO(3)(-), however, the outward current produced by D555E decreased only slightly after Cl(-) removal. Starting from a Cl(-)-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO(2)/HCO(3)(-). The substitution of Asp(555) with an asparagine also produced a Cl(-) current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO(3)(-). Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp(555) plays a role in HCO(3)(-) selectivity.

    Funded by: NIGMS NIH HHS: R01 GM078502

    The Journal of biological chemistry 2009;284;23;15970-9

  • NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway.

    Zhu Q, Azimov R, Kao L, Newman D, Liu W, Abuladze N, Pushkin A and Kurtz I

    Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1689, USA.

    The electrogenic Na(+)/HCO(3)(-) cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100 degrees arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong delta(-) helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a delta(-) helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.

    Funded by: NIDDK NIH HHS: DK058563, DK063125, DK077162; NIEHS NIH HHS: ES12935

    The Journal of biological chemistry 2009;284;13;8918-29

  • Sequence- or position-specific mutations in the carboxyl-terminal FL motif of the kidney sodium bicarbonate cotransporter (NBC1) disrupt its basolateral targeting and alpha-helical structure.

    Li HC, Collier JH, Shawki A, Rudra JS, Li EY, Mackenzie B and Soleimani M

    Center on Genetics of Transport, University of Cincinnati, Cincinnati, OH 45267, USA. Hong.Li@uc.edu

    The sodium-bicarbonate cotransporter NBC1 is targeted exclusively at the basolateral membrane. Mutagenesis of a dihydrophobic FL motif (residues 1013-1014) in the C-terminal domain disrupts the targeting of NBC1. In the present study, we determined the precise constraints of the FL motif required for basolateral targeting of NBC1 by expressing epitope-tagged wild-type and mutant NBC1 in MDCK cells and RNA-injected Xenopus oocytes and examining their subcellular localization. We assayed the functional activity of the mutants by measuring bicarbonate-induced currents in oocytes. Wild-type NBC1 (containing PFLS) was expressed exclusively on the basolateral membrane in MDCK cells. Reversal of the FL motif (PLFS) had no effect on basolateral targeting or activity. Shifting the FL motif one residue upstream (FLPS) resulted in mistargeting of the apical membrane but the FLPS mutant retained its functional activity in oocytes. Shifting the FL motif one residue downstream resulted in a mutant (PSFL) that did not efficiently translocate to the plasma membrane and was instead colocalized with the ER marker, protein disulfide isomerase (PDI). Analysis of circular dichroism (CD) revealed that a short peptide, 20 amino acid residues, of wild-type NBC1 contained a significant alpha-helical structure, whereas peptides in which the FL motif was reversed or C-terminally shifted were disordered. We therefore propose that the specific orientation and the precise location of the FL motif in the primary sequence of NBC1 are strict requirements for the alpha-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.

    Funded by: NIDDK NIH HHS: DK 62809, R01 DK080047

    The Journal of membrane biology 2009;228;2;111-24

  • cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells.

    Bachmann O, Franke K, Yu H, Riederer B, Li HC, Soleimani M, Manns MP and Seidler U

    Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany. bachmann.oliver@mh-hannover.de

    Background: The renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct.

    Results: Na+- and HCO3-dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10(-5) M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10(-4) M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na+/HCO3- cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue.

    Conclusion: Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.

    BMC cell biology 2008;9;70

  • G418-mediated ribosomal read-through of a nonsense mutation causing autosomal recessive proximal renal tubular acidosis.

    Azimov R, Abuladze N, Sassani P, Newman D, Kao L, Liu W, Orozco N, Ruchala P, Pushkin A and Kurtz I

    Division of Nephrology, David Geffen School of Medicine at UCLA, University of California-Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.

    Autosomal recessive proximal renal tubular acidosis is caused by mutations in the SLC4A4 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe1-A. The mutations that have been characterized thus far result in premature truncation, mistargeting, or decreased function of the cotransporter. Despite bicarbonate treatment to correct the metabolic acidosis, extrarenal manifestations persist, including glaucoma, cataracts, corneal opacification, and mental retardation. Currently, there are no known therapeutic approaches that can specifically target mutant NBCe1-A proteins. In the present study, we tested the hypothesis that the NBCe1-A-Q29X mutation can be rescued in vitro by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature stop codons. As a model system, we cloned the NBCe1-A-Q29X mutant into a vector lacking an aminoglycoside resistance gene and transfected the mutant cotransporter in HEK293-H cells. Cells transfected with the NBCe1-A-Q29X mutant failed to express the cotransporter because of the premature stop codon. Treatment of the cells with G418 significantly increased the expression of the full-length cotransporter, as assessed by immunoblot analysis. Furthermore, immunocytochemical studies demonstrated that G418 treatment induced cotransporter expression on the plasma membrane whereas in the absence of G418, NBCe1-A-Q29X was not expressed. In HEK293-H cells transfected with the NBCe1-A-Q29X mutant not treated with G418, NBCe1-A-mediated flux was not detectable. In contrast, in cells transfected with the NBCe1-A-Q29X mutant, G418 treatment induced Na(+)- and HCO(3)(-)-dependent transport that did not differ from wild-type NBCe1-A function. G418 treatment in mock-transfected cells was without effect. In conclusion, G418 induces ribosomal read-through of the NBCe1-A-Q29X mutation in HEK293-H cells. These findings represent the first evidence that in the presence of the NBCe1-A-Q29X mutation that causes proximal renal tubular acidosis, full-length functional NBCe1-A protein can be produced. Our results provide the first demonstration of a mutation in NBCe1-A that has been treated in a targeted and specific manner.

    Funded by: NIDDK NIH HHS: DK 058563, DK 063125, DK 077162, DK 07789

    American journal of physiology. Renal physiology 2008;295;3;F633-41

  • Entry to "formula tunnel" revealed by SLC4A4 human mutation and structural model.

    Chang MH, DiPiero J, Sönnichsen FD and Romero MF

    Department Physiology & Biophysics and Biology, Case Western Reserve University, Cleveland, OH 44106, USA.

    Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na(+) bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge-charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was "repaired" by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling HCO(3)(-) permeation.

    Funded by: NEI NIH HHS: EY 017732, R01 EY017732; NIDDK NIH HHS: DK 056218

    The Journal of biological chemistry 2008;283;26;18402-10

  • Familial pure proximal renal tubular acidosis--a clinical and genetic study.

    Katzir Z, Dinour D, Reznik-Wolf H, Nissenkorn A and Holtzman E

    Ze'ev Katzir, Pediatric Nephrology Services, E. Wolfson Medical Center, Holon, Israel. katzir@wolfson.health.gov.il

    Background: Inherited proximal renal tubular acidosis (pRTA) is commonly associated with more generalized proximal tubular dysfunctions and occasionally with other organ system defects. Inherited combined pRTA and distal RTA with osteopetrosis and pure pRTA associated with ocular abnormalities, a rare disease which has been recently described. Only one family with pure isolated pRTA has been reported so far and the genetic cause for this disease is unknown. Objectives. We report a unique family with isolated pRTA. The aim of the project was to define the phenotype and to try to find the gene defect causing the disease.

    Methods: Clinical and metabolic evaluation of all family members was performed and a family pedigree was constructed. DNA was extracted from blood samples of affected and unaffected family members. We amplified by PCR and sequenced the coding areas and splice-sites of the genes that contribute to HCO(-)(3) reclamation in the proximal tubule. The genes studied were as follows: CA II, CA IV, CA XIV, NCB1, Na(+)/H(+) exchanger (NHE)-3, NHE-8, the regulatory proteins of NHE3, NHRF1 and NHRF2 and the Cl(-)/HCO(-)(3) exchanger, SLC26A6.

    Results: The father and all four children had RTA with blood HCO(-)(3) levels of 11-14 meq/l and urine pH of 5.3-5.4. Increased HCO(-)(3) fractional excretion after bicarbonate loading to 40-60% confirmed the diagnosis pRTA. No other tubular dysfunction was found, and no organ system dysfunction was detected, besides short stature. No mutation was found in all candidate genes studied.

    Conclusions: We presented a second family in the literature with familial isolated pure pRTA. The mode of inheritance is compatible with an autosomal dominant disease. Because of the small size of the family, wide genome search was not applicable and the gene candidate approach was chosen. Nine important candidate genes were extensively studied but the molecular basis of the disease was not yet found and genotyping nine important gene candidates were negative.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2008;23;4;1211-5

  • Proximal RTA: Are all the charts completed yet?

    Gross P and Meye C

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2008;23;4;1101-2

  • Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis.

    Suzuki M, Vaisbich MH, Yamada H, Horita S, Li Y, Sekine T, Moriyama N, Igarashi T, Endo Y, Cardoso TP, de Sá LC, Koch VH, Seki G and Fujita T

    Department of Internal Medicine, Faculty of Medicine, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

    Mutations in the Na(+)-HCO(3)(-) cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

    Pflugers Archiv : European journal of physiology 2008;455;4;583-93

  • Identification of a novel signal in the cytoplasmic tail of the Na+:HCO3- cotransporter NBC1 that mediates basolateral targeting.

    Li HC, Li EY, Neumeier L, Conforti L and Soleimani M

    Dept. of Internal Medicine, Div. of Nephrology and Hypertension, Univ. of Cincinnati, 231 Albert Sabin Way, MSB G259, Cincinnati, OH 45267-0585, USA.

    The Na(+):HCO(3)(-) cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins.

    Funded by: NCI NIH HHS: CA-95286; NIDDK NIH HHS: DK-62809

    American journal of physiology. Renal physiology 2007;292;4;F1245-55

  • The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia.

    Toye AM, Parker MD, Daly CM, Lu J, Virkki LV, Pelletier MF and Boron WF

    Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.

    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.

    Funded by: NIDDK NIH HHS: DK-30344, R37 DK030344, R37 DK030344-28; NINDS NIH HHS: NS-18400; PHS HHS: P30-34989; Wellcome Trust

    American journal of physiology. Cell physiology 2006;291;4;C788-801

  • Role of NH(2) and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1.

    Espiritu DJ, Bernardo AA and Arruda JA

    Section of Nephrology, Department of Medicine, University of Illinois at Chicago, Chicago, Chicago, IL 60612-7378, USA.

    Sodium bicarbonate cotransporter 1 (NBC1) mediates 80% of bicarbonate reabsorption by the kidney, but the molecular determinants for activity, targeting, and cell membrane stability are poorly understood. We generated truncation mutants involving the entire NH(2) (DeltaN424) or the entire COOH (DeltaC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. DeltaN424 and DeltaC92 targeted to the plasma membrane of HEK293 cells or to the basolateral membrane of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the DeltaN424, DeltaC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total DeltaC92 expression in the cell was constant. We found that decreased DeltaC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both DeltaN424 and DeltaC92 were cotransfected together and more stable expression of DeltaC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH(2) epitope-specific antibody or vice versa. Similar findings were observed with Ni-NTA pull-down assay. Cotransfection of both mutants partially restored NBC1 activity. In summary, NBC1 targets to the basolateral membrane of OK cells by a default mechanism and the COOH terminus plays a role on NBC1 stability in the basolateral membrane.

    American journal of physiology. Renal physiology 2006;291;3;F588-96

  • Effect of human carbonic anhydrase II on the activity of the human electrogenic Na/HCO3 cotransporter NBCe1-A in Xenopus oocytes.

    Lu J, Daly CM, Parker MD, Gill HS, Piermarini PM, Pelletier MF and Boron WF

    Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. Jing.Lu@Yale.edu

    Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 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% CO2 (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.

    Funded by: NIDDK NIH HHS: DK-30344, F32 DK075258, K01 DK082646; PHS HHS: P30-34989

    The Journal of biological chemistry 2006;281;28;19241-50

  • Identification and membrane localization of electrogenic sodium bicarbonate cotransporters in Calu-3 cells.

    Kreindler JL, Peters KW, Frizzell RA and Bridges RJ

    Department of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA. james.kreindler@chp.edu

    Cystic fibrosis (CF) is a severely life-shortening genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). Impaired bicarbonate secretion is a key component of CF-related pancreatic disease, but the role of impaired bicarbonate secretion in CF lung disease is less well understood. The submucosal glands of the conducting airways produce and secrete a complex airway surface liquid that lines the airway epithelium and plays a significant role in mucociliary clearance. The serous cell is the predominant cell type of the submucosal gland and a predominant site of CFTR expression. Calu-3 cells are a model of airway submucosal gland serous cells that demonstrates vectorial bicarbonate secretion in response to elevations in cAMP. Based on previously published measurements of unidirectional ion flux, pharmacological inhibition of short-circuit current and ion substitution studies, one can hypothesize the existence of an electrogenic sodium bicarbonate cotransporter (NBC) in the basolateral membrane of Calu-3 cells that mediates bicarbonate entry from the interstitium. To test this hypothesis, we performed reverse-transcriptase PCR, western blotting, and surface biotinylation to identify and localize electrogenic NBCs in Calu-3 cells. Our data demonstrate that both pNBC1 and NBC4 mRNAs can be identified and that their protein products are expressed at the basolateral membrane of polarized Calu-3 cells. These data suggest that these transporters contribute to regulated bicarbonate secretion across Calu-3 cells and perhaps human airway submucosal glands.

    Biochimica et biophysica acta 2006;1762;7;704-10

  • PMA- and ANG II-induced PKC regulation of the renal Na+-HCO3- cotransporter (hkNBCe1).

    Perry C, Blaine J, Le H and Grichtchenko II

    Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Mail Stop 8307, P.O. Box 6511, Aurora, CO 80045, USA.

    The renal electrogenic Na(+)-HCO3- cotransporter (hkNBCe1) plays a major role in the bicarbonate reabsorption by the kidney. We examined how PMA- and ANG II-activated PKCs regulate hkNBCe1 expressed with or without the ANG II receptors AT(1B) in Xenopus laevis oocytes. We found that 10 nM PMA halved the hkNBCe1 current detected in voltage-clamped oocytes. A PKC-specific inhibitor GF-109203X, and a specific inhibitor of Ca-dependent conventional PKCalphabetagamma, GO-6976, significantly reduced PMA inhibition. PMA did not alter surface expression of the cotransporters, but it significantly increased hkNBCe1-PKCalphabetagamma membrane association. We found that at 10(-6) M, ANG II halved the hkNBCe1 current detected in oocytes coexpressing cotransporters with AT(1B). A PKC-specific inhibitor GF-109203X, and a PKCepsilon translocation inhibitor epsilonV1-2 peptide as well as BAPTA-AM (but not GO-6976), significantly reduced ANG II inhibition. At 10(-6) M, ANG II significantly decreased surface expression of the cotransporters and increased hkNBCe1-PKCepsilon membrane association. Additionally, we found that at 10(-11) and 10(-10) M ANG II stimulated hkNBCe1 current. This effect was blocked by BAPTA-AM and partially reduced by GF-109203X. We also found that ANG II increased intracellular Ca2+ in fluo 4-loaded oocytes. Our results suggest that 1) PMA inhibition of hkNBCe1 is mediated by Ca-dependent PKCalphabetagamma and 10 nM PMA does not induce downregulation of cotransporter surface expression. 2) ANG II (10(-6) M) inhibition of hkNBCe1 is mediated by both Ca-independent PKCepsilon and downregulation of cotransporter surface expression, possibly triggered by intracellular Ca2+ mobilization. 3) Similar to proximal tubule, acute ANG II has a biphasic effect on hkNBCe1 coexpressed with AT(1B) in X. laevis oocytes.

    American journal of physiology. Renal physiology 2006;290;2;F417-27

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Functional analysis of NBC1 mutants associated with proximal renal tubular acidosis and ocular abnormalities.

    Horita S, Yamada H, Inatomi J, Moriyama N, Sekine T, Igarashi T, Endo Y, Dasouki M, Ekim M, Al-Gazali L, Shimadzu M, Seki G and Fujita T

    Department of Internal Medicine, Faculty of Medicine, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

    Mutations in the Na+-HCO3- co-transporter (NBC1) cause permanent proximal renal tubular acidosis (pRTA) with ocular abnormalities. However, little has been known about the relationship between the degree of NBC1 inactivation and the severity of pRTA. This study identified three new homozygous mutations (T485S, A799V, and R881C) in the common coding regions of NBC1. Functional analysis of these new as well as the known mutants (R298S and R510H) in Xenopus oocytes revealed a considerable variation in their electrogenic activities. Whereas the activities of R298S, A799V, and R881C were 15 to 40% of the wild-type (WT) activity, T485S and R510H, as a result of poor surface expression, showed almost no activities. However, T485S, like R510H, had the transport activity corresponding to approximately 50% of the WT activity in ECV304 cells, indicating that surface expression of T485S and R510H varies between the different in vitro cell systems. Electrophysiologic analysis showed that WT, R298S, and R881C all function with 2HCO3- to 1Na+ stoichiometry and have similar extracellular Na+ affinity, indicating that reduction in Na+ affinity cannot explain the inactivation of R298S and R881C. These results, together with the presence of nonfunctional mutants (Q29X and DeltaA) in other patients, suggest that at least approximately 50% reduction of NBC1 activity would be required to cause severe pRTA.

    Journal of the American Society of Nephrology : JASN 2005;16;8;2270-8

  • Missense mutations in Na+:HCO3- cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis.

    Li HC, Szigligeti P, Worrell RT, Matthews JB, Conforti L and Soleimani M

    Dept. of Medicine, University of Cincinnati, Cincinnati, OH 45267-0585, USA.

    The kidney Na(+):HCO(3)(-) cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH(2) terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.

    Funded by: NCI NIH HHS: CA-095286, R01 CA095286-03; NIDDK NIH HHS: DK-51630, DK-62809, R01 DK051630-09, R01 DK062809-02

    American journal of physiology. Renal physiology 2005;289;1;F61-71

  • Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport.

    Abuladze N, Azimov R, Newman D, Sassani P, Liu W, Tatishchev S, Pushkin A and Kurtz I

    Division of Nephrology, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Room 7-155 Factor Building, Los Angeles, CA 90095, USA.

    We have previously reported a topological model of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na(+)-dependent base flux of the mutants was determined using intracellular pH measurements with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na(+)-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented.

    Funded by: NIDDK NIH HHS: DK07789, DK58563, DK63125, R01 DK058563, R01 DK063125, T32 DK007789

    The Journal of physiology 2005;565;Pt 3;717-30

  • Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration.

    Yang Z, Alvarez BV, Chakarova C, Jiang L, Karan G, Frederick JM, Zhao Y, Sauvé Y, Li X, Zrenner E, Wissinger B, Hollander AI, Katz B, Baehr W, Cremers FP, Casey JR, Bhattacharya SS and Zhang K

    Department of Ophthalmology and Visual Science, University of Utah Health Science Center, Salt Lake City 84112, USA.

    Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown. Here, we show that a functional complex of carbonic anhydrase 4 (CA4) and Na+/bicarbonate co-transporter 1 (NBC1) is specifically expressed in the choriocapillaris and that missense mutations in CA4 linked to autosomal dominant rod-cone dystrophy disrupt NBC1-mediated HCO3- transport. Our results identify a novel pathogenic pathway in which a defect in a functional complex involved in maintaining pH balances, but not expressed in retina or RPE, leads to photoreceptor degeneration. The importance of a functional CA4 for survival of photoreceptors implies that CA inhibitors, which are widely used as medications, particularly in the treatment of glaucoma, may have long-term adverse effects on vision.

    Funded by: NCRR NIH HHS: M01-RR00064; NEI NIH HHS: EY08123, R01EY14428, R01EY14448

    Human molecular genetics 2005;14;2;255-65

  • A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects.

    Dinour D, Chang MH, Satoh J, Smith BL, Angle N, Knecht A, Serban I, Holtzman EJ and Romero MF

    Department of Nephrology and Hypertension, Chaim Sheba Medical Center, Tel-Hashomer, and Tel-Aviv University, 52621 Israel.

    In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na+/HCO3- cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264-266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO3- transport) and membrane currents using microelectrodes. HCO3- -elicited currents for S427L were approximately 10% of WT NBCe1, and CO2-induced acidification was approximately 4-fold faster. Na+ -dependent HCO3- transport (currents and acidification) was also approximately 10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO3-, indicating that under physiological ion gradient conditions, NaHCO3 could not move out of cells as is needed for renal HCO3- absorption and ocular pressure homeostasis. I-V analysis without Na+ further shows that the S427L-mediated NaHCO3 efflux mode is depressed or absent. These experiments reveal that voltage- and Na+ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO3- absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).

    Funded by: NIDDK NIH HHS: DK-56218

    The Journal of biological chemistry 2004;279;50;52238-46

  • Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane.

    Li HC, Worrell RT, Matthews JB, Husseinzadeh H, Neumeier L, Petrovic S, Conforti L and Soleimani M

    Department of Medicine, University of Cincinnati, Cincinnati, Ohio 45267, USA.

    The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.

    Funded by: NCI NIH HHS: CA095286; NIDDK NIH HHS: DK 51630, DK 62809

    The Journal of biological chemistry 2004;279;41;43190-7

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Expression of Na+/HCO3- co-transporter proteins (NBCs) in rat and human skeletal muscle.

    Kristensen JM, Kristensen M and Juel C

    Copenhagen Muscle Research Centre, August Krogh Institute, University of Copenhagen, Denmark.

    Aim: Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs.

    Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions.

    Conclusions: Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2.

    Acta physiologica Scandinavica 2004;182;1;69-76

  • Molecular mechanism of kNBC1-carbonic anhydrase II interaction in proximal tubule cells.

    Pushkin A, Abuladze N, Gross E, Newman D, Tatishchev S, Lee I, Fedotoff O, Bondar G, Azimov R, Ngyuen M and Kurtz I

    Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. apushkin@mednet.ucla.edu

    We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L(958)DDV and D(986)NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.

    Funded by: NIDDK NIH HHS: DK07789, DK58563, DK63125, R01 DK058563, R01 DK063125, T32 DK007789

    The Journal of physiology 2004;559;Pt 1;55-65

  • Voltage dependence of H+ buffering mediated by sodium bicarbonate cotransport expressed in Xenopus oocytes.

    Becker HM and Deitmer JW

    Abteilung für Allgemeine Zoologie, Fachbereich Biologie, Technische Universität Kaiserslautern, D-67653 Kaiserslautern, Germany.

    The electrogenic sodium bicarbonate cotransporter (NBCe1) is expressed in many epithelial cells and, in the brain, in glial cells. Little is known about the physiological significance of the NBCe1 for proton homeostasis and for other acid/base-coupled transporters in these cells. We have measured the voltage-dependent transport activity of an NBC from human kidney, type hkNBCe1, expressed in oocytes of the frog Xenopus laevis, by recording membrane current and the changes in intracellular pH and sodium at different membrane potentials between -20 and -100 mV. The apparent intracellular buffer capacity was increased and became dependent upon membrane voltage when the NBCe1 was expressed; the measured buffer capacity increased by up to 7 mm/10 mV of membrane depolarization. Lactate transport by the electroneutral monocarboxylate transporter became enhanced and dependent upon membrane potential, when the monocarboxylate transporter (isoform 1) was co-expressed with NBCe1 in oocytes. Our results indicate that the electrogenic NBCe1 renders the cell membrane potential an effective regulator of intracellular H(+) buffering and acid/base-coupled metabolite transport.

    The Journal of biological chemistry 2004;279;27;28057-62

  • Regulation of renal Na+/HCO3- cotransporter stimulation by CO2: role of phosphorylation, exocytosis and protein synthesis.

    Espiritu DJ, Yang VL, Bernardo AA and Arruda JA

    Section of Nephrology, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612-7315, USA.

    The sodium bicarbonate cotransporter (NBC1) mediates bicarbonate reabsorption in the renal proximal tubule. NBC1 activity is stimulated by 10% CO2, however, the mechanism is poorly understood. Here, we examined the mechanism of NBC1 regulation by 10% CO2 using an immortalized human proximal tubule cell line (HK2). In cells exposed to 10% CO2, the cotransporter activity (measured as deltapH/min) increased within minutes and this increase was maintained for 6 to 24 h. Early NBC1 stimulation was accompanied by increased NBC1 phosphorylation. Basolateral membrane NBC1 protein increased by 30 min and reached a maximum at 6 h. Increased NBC activity at 6 h was accounted for by increased NBC exocytosis to the basolateral membrane and not by decreased endocytosis. Latruncullin B (an actin cytoskeleton inhibitor) did not prevent CO2-induced stimulation, while nocodazole (a microtubule-disrupting agent) abrogated the stimulatory effect of 10% CO2. A significant increase in NBC1 mRNA expression level was observed at 6 h and maintained for 24 h. Total NBC1 protein increased at 12 to 24 h with 10% CO2 incubation and this effect was blocked by cycloheximide. In summary, the present study demonstrates that early activation of NBC1 activity by 10% CO2 was mediated by NBC1 phosphorylation. The stimulation of cotransporter activity observed at 6 h was due to exocytosis, while the late effect starting from 12 h was accounted for by increased protein synthesis.

    The Journal of membrane biology 2004;199;1;39-49

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Localization of NBC-1 variants in human kidney and renal cell carcinoma.

    Yamada H, Yamazaki S, Moriyama N, Hara C, Horita S, Enomoto Y, Kudo A, Kawakami H, Tanaka Y, Fujita T and Seki G

    Department of Internal Medicine, Faculty of Medicine, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

    The Na(+)-HCO(3)(-) cotransporter (NBC-1) plays a major role in bicarbonate absorption from proximal tubules. However, which NBC-1 variant mediates proximal bicarbonate absorption has not been definitely determined. Moreover, the localization of this cotransporter in human kidney and renal cell carcinoma (RCC) tissues has not been clarified. To clarify these issues, immunohistochemical analysis was performed using the specific antibodies against kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters. In Western blot analysis the expression of kNBC-1 but not of pNBC-1 was detected in both normal human kidney and RCC tissues. In immunofluorescence analysis on normal renal tissues the anti-kNBC-1 antibody strongly and exclusively labeled the basolateral membranes of proximal tubules, which was confirmed by electron microscopic observation. In RCC cells, the anti-kNBC-1 antibody labeled both plasma membranes and intracellular organelles. The labeling by anti-pNBC-1 antibody was not detected in both normal kidney and RCC tissues. These results indicate that kNBC-1 is the dominant variant that mediates bicarbonate absorption from human renal proximal tubules. They also suggest that NBC-1 may have distinct roles in cancer cells.

    Biochemical and biophysical research communications 2003;310;4;1213-8

  • Direct extracellular interaction between carbonic anhydrase IV and the human NBC1 sodium/bicarbonate co-transporter.

    Alvarez BV, Loiselle FB, Supuran CT, Schwartz GJ and Casey JR

    Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

    Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pH(i)) and HCO(3)(-) metabolism. Electrogenic NBC1 catalyzes HCO(3)(-) fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydration-dehydration of CO(2)/HCO(3)(-). The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pH(i) in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pH(i) recovery by 44 +/- 3%, as compared to NBC1-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAIV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAIV interaction. NBC1-mediated pH(i) recovery rate after acid load was inhibited by 40 +/- 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAII competes with endogenous CAII for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full pH(i) recovery activity. We conclude that CAIV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAII and CAIV close to the NBC1 HCO(3)(-) transport site maximizes the transmembrane HCO(3)(-) gradient local to NBC1 and thereby activates the transport rate.

    Funded by: NIDDK NIH HHS: DK-50603

    Biochemistry 2003;42;42;12321-9

  • Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.

    Sun XC and Bonanno JA

    School of Optometry, Indiana University, 800 E. Atwater Ave. Bloomington, IN 47401, USA. jbonanno@indiana.edu

    Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.

    Funded by: NEI NIH HHS: EY08834, R01 EY008834, R24 EY016661

    Experimental eye research 2003;77;3;287-95

  • Phosphorylation-induced modulation of pNBC1 function: distinct roles for the amino- and carboxy-termini.

    Gross E, Fedotoff O, Pushkin A, Abuladze N, Newman D and Kurtz I

    Departments of Reproductive Biology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. ezg@po.cwru.edu

    The human NBC1 (SLC4A4) gene encodes the electrogenic sodium bicarbonate cotransporters kNBC1 and pNBC1, which are highly expressed in the kidney and pancreas, respectively. The HCO3-:Na+ stoichiometry of these cotransporters is an important determinant of the direction of ion flux. Recently we showed in a mouse proximal tubule (mPCT) cell line expressing kNBC1, that 8-Br-cAMP shifts the stoichiometry of the cotransporter from 3:1 to 2:1 via protein kinase A (PKA)-dependent phosphorylation of Ser982. pNBC1 has the identical carboxy-terminal consensus phosphorylation PKA site (KKGS1026), and an additional site in its amino-terminus (KRKT49). In this study we determined the potential role of these sites in regulating the function of pNBC1. The results demonstrated that in mPCT cells expressing pNBC1, PKA-dependent phosphorylation of Ser1026 following 8-Br-cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser1026 with Asp resulted in a similar stoichiometry shift. In addition to shifting the stoichiometry, 8-Br-cAMP caused a significant increase in the 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive basolateral membrane conductance (GDS) of cells expressing pNBC1, but not kNBC1. Although, the effect did not involve phosphorylation of Thr49, which was endogenously phosphorylated, replacing this residue with Asp or Ala abolished the 8-Br-cAMP-induced increase in GDS. In the mPEC pancreatic duct cell line, where endogenous pNBC1 functions with a HCO3-:Na+ stoichiometry of 2:1, 8-Br-cAMP increased GDS by ~90 % without altering the stoichiometry or inducing phosphorylation of the cotransporter. The results demonstrate that phosphorylation of Ser1026 mediates the cAMP-dependent shift in the stoichiometry of pNBC1, whereas Thr49 plays an essential role in the cAMP-induced increase in GDS.

    Funded by: NIDDK NIH HHS: DK-07789, DK-58563, DK-63125, R01 DK058563, R01 DK063125, T32 DK007789

    The Journal of physiology 2003;549;Pt 3;673-82

  • Identification of membrane topography of the electrogenic sodium bicarbonate cotransporter pNBC1 by in vitro transcription/translation.

    Tatishchev S, Abuladze N, Pushkin A, Newman D, Liu W, Weeks D, Sachs G and Kurtz I

    Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.

    The transmembrane topography of the human pancreatic electrogenic sodium bicarbonate cotransporter pNBC1 was investigated using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of pNBC1 hydrophobic sequences (H). These vectors encode N-terminal 101 (HK-M0) or 139 (HK-M1) amino acids of the H,K-ATPase alpha-subunit, a linker region and the C-terminal 177 amino acids of the H,K-ATPase beta-subunit that contain five N-linked glycosylation consensus sites (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). The glycosylation status of the beta-region was used as a reporter to determine whether a given hydrophobic sequence possesses signal anchor and/or stop transfer properties in the HK-M0 and HK-M1 vectors. The linker region of each vector was replaced either with individual hydrophobic sequences or combinations thereof. The transcription/translation products of these fusion vectors in reticulocyte lysate system +/- microsomal membranes were identified by [(35)S]-autoradiography following separation using SDS-PAGE. The results of the in vitro transcription/translation analysis indicated that 10 (H1, H2N, H3, H5, H6, H7, H8, H9, H11, and H12) out of 12 hydrophobic sequences were able to insert into the plasma membrane. Two hydrophobic sequences, H4 and H10, had no membrane insertion activity even when upstream and downstream sequences were present. These data and immunocytochemical studies indicate that pNBC1 contains 10 transmembrane domains with N- and C-termini oriented intracellularly. This is the first characterization of the membrane topography of a sodium bicarbonate cotransporter.

    Funded by: NIDDK NIH HHS: DK-07789, DK-58563

    Biochemistry 2003;42;3;755-65

  • Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp(986), Asp(988) and kNBC1-carbonic anhydrase II binding.

    Gross E, Pushkin A, Abuladze N, Fedotoff O and Kurtz I

    Department of Urology, Case Western Reserve University, Cleveland, OH, USA. ezg@po.cwru.edu

    The HCO(3)(-) : Na(+) cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (E(rev)) and thus the net direction of transport of these ions through the cotransporter. Previously, we showed that phosphorylation of kNBC1-Ser(982) in the carboxy-terminus of kNBC1 (kNBC1-Ct), by cAMP-protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the cotransporter is electrostaticaly modulated. These results raise the possibility that phosphorylated kNBC1-Ser(982), or other nearby negatively charged residues shift the stoichiometry by blocking a bicarbonate-binding site. In the current study, we examined the role of the negative charge on Ser(982)-phosphate and three aspartate residues in a D986NDD custer in altering the stoichiometry of kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in an Ussing chamber for electrophysiological studies. Enhanced green fluorescence protein (EGFP)-tagged mutant constructs expressed in the same cells were used to determine the phosphorylation status of kNBC1-Ser(982). The data indicate that both kNBC1-Asp(986) and kNBC1-Asp(988), but not kNBC1-Asp(989), are required for the phosphorylation-induced shift in stoichiometry. A homologous motif (D887ADD) in the carboxy-terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). In isothermal titration calorimetry experiments, CAII was found to bind to kNBC1-Ct with a K(D) of 160 +/- 10 nM. Acetazolamide inhibited the short-circuit current through the cotransporter by 65 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. Acetazolamide did not affect the cotransport stoichiometry or the ability of 8-Br-cAMP to shift the stoichiometry. Although CAII does not affect the transport stoichiometry, it may play an important role in enhancing the flux through the transporter when kNBC1-Ser(982) is unphosphorylated.

    Funded by: NIDDK NIH HHS: DK07789, DK58563, R01 DK058563, T32 DK007789

    The Journal of physiology 2002;544;3;679-85

  • Expression of a sodium bicarbonate cotransporter in human parotid salivary glands.

    Park K, Hurley PT, Roussa E, Cooper GJ, Smith CP, Thévenod F, Steward MC and Case RM

    School of Biological Sciences, University of Manchester, G.38 Stopford Building, M13 9PT, Manchester, UK.

    The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.

    Archives of oral biology 2002;47;1;1-9

  • Phosphorylation of Ser(982) in the sodium bicarbonate cotransporter kNBC1 shifts the HCO(3)(-) : Na(+) stoichiometry from 3 : 1 to 2 : 1 in murine proximal tubule cells.

    Gross E, Hawkins K, Pushkin A, Sassani P, Dukkipati R, Abuladze N, Hopfer U and Kurtz I

    Department of Urology, Case Western Reserve University and VA Medical Center, Cleveland, OH 44106, USA. ezg@po.cwru.edu

    1. Adenosine 3',5'-cyclic monophosphate (cAMP) modulates proximal tubule sodium and bicarbonate absorption by decreasing the rate of apical Na(+)-H(+) exchange and basolateral sodium bicarbonate efflux, through activation of protein kinase A (PKA). The electrogenic sodium bicarbonate cotransporter kNBC1 mediates basolateral sodium and bicarbonate efflux in the proximal tubule by coupling the transport of 1 Na(+) cation to that of 3 HCO(3)(-) anions. In this work we studied the effects of cAMP on the function of kNBC1 expressed heterologously in a proximal tubule cell line. 2. A mouse renal proximal tubule cell line, deficient in electrogenic sodium bicarbonate cotransport function, was transfected with kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The HCO(3)(-) : Na(+) stoichiometry of kNBC1 was calculated from its reversal potential by measuring the current-voltage relationships of the cotransporter at different Na(+) concentration gradients. 3. Addition of the potent cAMP agonist 8-Br-cAMP caused the stoichiometry of kNBC1 to shift from 3 HCO(3)(-) : 1 Na(+) to 2 HCO(3)(-) : 1 Na(+). Pretreatment of the cells with the PKA inhibitor H-89 abolished the effect of the agonist on the stoichiometry change. Replacing Ser(982) at the C-terminus consensus PKA phosphorylation site with alanine resulted in a failure of PKA to phosphorylate the transporter and induce a stoichiometry shift. 4. Our data indicate that cAMP modulates the stoichiometry of kNBC1 through activation of PKA. The change in stoichiometry from 3 : 1 to 2 : 1 is predicted to cause a shift in the direction of basolateral membrane sodium bicarbonate transport from efflux to influx. Ser(982) in the C-terminus of kNBC1 is a target for PKA phosphorylation. This is the first example of modulation of the stoichiometry of a membrane transporter by phosphorylation.

    Funded by: NHLBI NIH HHS: HL-41618, P01 HL041618; NIDDK NIH HHS: DK46976, DK54221, DK6976, R01 DK054221

    The Journal of physiology 2001;537;Pt 3;659-65

  • Physiologic and molecular aspects of the Na+:HCO3- cotransporter in health and disease processes.

    Soleimani M and Burnham CE

    Department of Medicine, University of Cincinnati, and the Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585, USA. Manoocher.Soleimani@uc.edu

    Approximately 80% of the filtered load of HCO3- is reabsorbed in the proximal tubule via a process of active acid secretion by the luminal membrane. The major mechanism for the transport of HCO3- across the basolateral membrane is via the electrogenic Na+:3HCO3- cotransporter (NBC). Recent molecular cloning experiments have identified the existence of three NBC isoforms (NBC-1, NBC-2, and NBC-3).1 Functional and molecular studies indicate the presence of all three NBC isoforms in the kidney. All are presumed to mediate the cotransport of Na+ and HCO3- under normal conditions and may be functionally altered in certain pathophysiologic states. Specifically, NBC-1 may be up-regulated in metabolic acidosis and potassium depletion and in response to glucocorticoid excess and may be down-regulated in response to HCO3- loading or alkalosis. Recent studies provide molecular evidence indicating the expression of NBC-1 in pancreatic duct cells. NBC is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO3- secretion in the agonist-stimulated state in pancreatic duct cells. The purpose of this review is to summarize recent functional and molecular studies on the regulation of NBCs in physiologic and pathophysiologic states. Possible signals responsible for the regulation of NBCs in these conditions are examined. Furthermore, the possible role of this transporter in acid-base disorders (such as proximal renal tubular acidosis) is discussed.

    Funded by: NIDDK NIH HHS: R01 DK52821, R01 DK54430, R01DK 46789

    Kidney international 2000;57;2;371-84

  • NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase.

    Pushkin A, Yip KP, Clark I, Abuladze N, Kwon TH, Tsuruoka S, Schwartz GJ, Nielsen S and Kurtz I

    Division of Nephrology, University of California at Los Angeles, School of Medicine, Los Angeles, California 90095, USA.

    We have recently cloned and characterized a unique sodium bicarbonate cotransporter, NBC3, which unlike other members of the NBC family, is ethylisopropylamiloride (EIPA) inhibitable, DIDS insensitive, and electroneutral (A. Pushkin, N. Abuladze, I. Lee, D. Newman, J. Hwang, and I. Kurtz. J. Biol. Chem. 274: 16569-16575, 1999). In the present study, a specific polyclonal antipeptide COOH-terminal antibody, NBC3-C1, was generated and used to determine the pattern of NBC3 protein expression in rabbit kidney. A major band of approximately 200 kDa was detected on immunoblots of rabbit kidney. Immunocytochemistry of rabbit kidney frozen sections revealed specific staining of the apical membrane of intercalated cells in both the cortical and outer medullary collecting ducts. The pattern of NBC3 protein expression in the collecting duct was nearly identical to the same sections stained with an antibody against the vacuolar H+-ATPase 31-kDa subunit. In addition, the NBC3-C1 antibody coimmunoprecipitated the vacuolar H+-ATPase 31-kDa subunit. Functional studies in outer medullary collecting ducts (inner stripe) showed that type A intercalated cells have an apical Na+-dependent base transporter that is EIPA inhibitable and DIDS insensitive. The data suggest that NBC3 participates in H+/base transport in the collecting duct. The close association of NBC3 and the vacuolar H+-ATPase in type A intercalated cells suggests a potential structural/functional interaction between the two transporters.

    Funded by: NHLBI NIH HHS: HL-59156; NIDDK NIH HHS: DK-46976, DK-50603

    The American journal of physiology 1999;277;6;F974-81

  • Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities.

    Igarashi T, Inatomi J, Sekine T, Cha SH, Kanai Y, Kunimi M, Tsukamoto K, Satoh H, Shimadzu M, Tozawa F, Mori T, Shiobara M, Seki G and Endou H

    Nature genetics 1999;23;3;264-6

  • Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC).

    Choi I, Romero MF, Khandoudi N, Bril A and Boron WF

    Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. inyeong.choi@qm.yale.edu

    Our group recently cloned the electrogenic Na+-HCO-3 cotransporter (NBC) from salamander kidney and later from mammalian kidney. Here we report cloning an NBC isoform (hhNBC) from a human heart cDNA library. hhNBC is identical to human renal NBC (hkNBC), except for the amino terminus, where the first 85 amino acids in hhNBC replace the first 41 amino acids of hkNBC. About 50% of the amino acid residues in this unique amino terminus are charged, compared with approximately 22% for the corresponding 41 residues in hkNBC. Northern blot analysis, with the use of the unique 5' fragment of hhNBC as a probe, shows strong expression in pancreas and expression in heart and brain, although at much lower levels. In Xenopus oocytes expressing hhNBC, adding 1.5% CO2/10 mM HCO-3 hyperpolarizes the membrane and causes a rapid fall in intracellular pH (pHi), followed by a pHi recovery. Subsequent removal of Na+ causes a depolarization and a reduced rate of pHi recovery. Removal of Cl- from the bath does not affect the pHi recovery. The stilbene derivative DIDS (200 microM) greatly reduces the hyperpolarization caused by adding CO2/HCO-3. In oocytes expressing hkNBC, the effects of adding CO2/HCO-3 and then removing Na+ were similar to those observed in oocytes expressing hhNBC. We conclude that hhNBC is an electrogenic Na+-HCO-3 cotransporter and that hkNBC is also electrogenic.

    Funded by: NIDDK NIH HHS: DK-09342, DK-30344, F32 DK009342, F32 DK009342-01, F32 DK009342-02, F32 DK009342-03

    The American journal of physiology 1999;276;3;C576-84

  • Electrogenic Na+/HCO3- cotransporters: cloning and physiology.

    Romero MF and Boron WF

    Department of Physiology and Biophysics and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4790, USA. mfr2@po.cwru.edu

    Bicarbonate and CO2 comprise the major pH buffer of biological fluids. In the renal proximal tubule most of the filtered HCO3- is reabsorbed by an electrogenic Na/HCO3 cotransporter located at the basolateral membrane. This Na+ bicarbonate cotransporter (NBC) was recently cloned. This review highlights the recent developments leading to and since the cloning of NBC: NBC expression cloning, protein features, clone physiology, isoforms and genes, mRNA distribution, and protein distribution. With the NBC amino acid sequence 30-35% identical to the anion exchangers (AE1-3), a superfamily of HCO3- transporters is emerging. Physiologically, NBC is electrogenic, Na+ dependent, HCO3- dependent, Cl- independent, and inhibited by stilbenes (DIDS and SITS). NBC clones and proteins have been isolated from several tissues (other than kidney) thought to have physiologically distinct HCO3- transporters. For example, NBC occurs in pancreas, prostate, brain, heart, small and large intestine, stomach, and epididymis. Finally, there are at least two genes that encode NBC proteins. Possible future directions of research are discussed.

    Funded by: NIDDK NIH HHS: DK17433, DK30344, DK43171, F32 DK009342, F32 DK009342-01, F32 DK009342-02, F32 DK009342-03

    Annual review of physiology 1999;61;699-723

  • Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter.

    Abuladze N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A and Kurtz I

    Division of Nephrology, Center for Health Sciences, UCLA School of Medicine, Los Angeles, California 90095-1698, USA.

    We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem. 272, 19111-19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an approximately 7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5' region of kNBC detected an approximately 7.6-kilobase transcript only in the kidney. In situ hybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescent in situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney.

    Funded by: NIDDK NIH HHS: DK46976

    The Journal of biological chemistry 1998;273;28;17689-95

  • Cloning and functional expression of a human kidney Na+:HCO3- cotransporter.

    Burnham CE, Amlal H, Wang Z, Shull GE and Soleimani M

    Department of Internal Medicine, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267-0585, USA.

    Several modes of HCO3- transport occur in the kidney, including Na+-independent Cl/HCO3- exchange (mediated by the AE family of Cl-/HCO3- exchangers), sodium-dependent Cl-/HCO3- exchange, and Na+:HCO3- cotransport. The functional similarities between the Na+-coupled HCO3- transporters and the AE isoforms (i.e. transport of HCO3- and sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) suggested a strategy for cloning the other transporters based on structural similarity with the AE family. An expressed sequence tag encoding part of a protein that is related to the known anion exchangers was identified in the GenBankTM expressed sequence tag data base and used to design an oligonucleotide probe. This probe was used to screen a human kidney cDNA library. Several clones were identified, isolated, and sequenced. Two overlapping cDNA clones were spliced together to form a 7.6-kilobase cDNA that contained the entire coding region of a novel protein. Based on the deduced amino acid sequence, the cDNA encodes a protein with a Mr of 116,040. The protein has 29% identity with human brain AE3. Northern blot analysis reveals that the 7.6-kilobase mRNA is highly expressed in kidney and pancreas, with detectable levels in brain. Functional studies in transiently transfected HEK-293 cells demonstrate that the cloned transporter mediates Na+:HCO3- cotransport.

    Funded by: NIDDK NIH HHS: DK 46789, DK 50594

    The Journal of biological chemistry 1997;272;31;19111-4

  • 3,400 new expressed sequence tags identify diversity of transcripts in human brain.

    Adams MD, Kerlavage AR, Fields C and Venter JC

    Receptor Biochemistry and Molecular Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.

    We present the results of the partial sequencing of over 3,400 expressed sequence tags (ESTs) from human brain cDNA clones, which increases the number of distinct genes expressed in the brain, that are represented by ESTs, to about 6,000. By choosing clones in an unbiased manner, it is possible to construct a profile of the transcriptional activity of the brain at different stages. Proteins that comprise the cytoskeleton are the most abundant; however, a large variety of regulatory proteins are also seen. About half of the ESTs predicted to contain a protein-coding region have no matches in the public peptide databases and may represent new gene families.

    Nature genetics 1993;4;3;256-67

Gene lists (7)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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