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WO1998053067A1 - Le nbc, gene codant pour un membre la famille de proteines transporteuses de bicarbonate - Google Patents

Le nbc, gene codant pour un membre la famille de proteines transporteuses de bicarbonate Download PDF

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Publication number
WO1998053067A1
WO1998053067A1 PCT/US1998/010297 US9810297W WO9853067A1 WO 1998053067 A1 WO1998053067 A1 WO 1998053067A1 US 9810297 W US9810297 W US 9810297W WO 9853067 A1 WO9853067 A1 WO 9853067A1
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Prior art keywords
nbc
protein
hco
nucleic acid
proteins
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PCT/US1998/010297
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English (en)
Inventor
Mark O. Bevensee
Bernhard M. Schmitt
Michael F. Romero
Walter F. Boron
Daniel Biemesderfer
Bruce A. Davis
Caroline R. Sussman
Inyeong Choi
Christian Aalkjaer
Irina I. Grichtchenko
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Yale University
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Priority to AU76892/98A priority Critical patent/AU7689298A/en
Publication of WO1998053067A1 publication Critical patent/WO1998053067A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians

Definitions

  • the present invention relates to the fields of extra and intracellular pH and cellular transport of sodium ions (Na + ) , bicarbonate ions (HCON) and water.
  • the invention relates specifically to the identification of a novel gene, tentatively named NBC (Na, Bicarbonate Cotransporter).
  • NBC encodes a protein, NBC, that is a member of the Bicarbonate Transporter family of proteins.
  • the "pumps” in this case are acid extruders (i.e., active transporters that tend to increase pHj), such as the vacuolar-type H + pump and the Na-H exchanger.
  • the "leaks” are acid loaders, passive mechanisms that tend to decrease pHj.
  • HCO ⁇ 3 One of the major functions of the renal proximal tubule is to reclaim HCO ⁇ 3 that has been filtered in the glomerulus.
  • This reabsorption of HCO ⁇ 3 from the proximal-tubule lumen to the blood helps to maintain an appropriately high [HCO " 3 ] in the blood, and thus helps to stabilize blood pH. Failure to reabsorb sufficient HCO 3 would not only lead to the loss of HCO ⁇ 3 m m e urine, but also to the loss of Na + (which normally accompanies reabsorbed HCO ⁇ 3 ) and osmotically obligated water. Thus, the consequences of reduced Na reabsorption would be metabolic acidosis and volume depletion.
  • HCO ⁇ 3 reabsorption by the proximal tubule is a multistep process.
  • H + is secreted into the lumen by Na + /H + exchangers (antiporters) and presumably vacuolar-type H + pumps (V-type ATPases).
  • this H + titrates luminal HCO ⁇ 3 to CO 2 and H 2 O, a process accelerated by carbonic anhydrase (CA) IV, which is tethered to the extracellular surface of the apical membrane.
  • CA carbonic anhydrase
  • the newly formed CO 2 and H 2 O enter the proximal-tubule cell.
  • cytoplasmic CA II accelerates the regeneration of H + and HCO ⁇ 3 .
  • this HCO ⁇ 3 exits the proximal-tubule cell across the basolateral membrane (BLM), completing the movement of HCO ⁇ 3 from lumen to blood.
  • the efflux of HCO ⁇ 3 across the basolateral membrane is mediated both by a Cl ⁇ HCO ⁇ 3 exchanger and an electrogenic Na + :HCO ⁇ 3 cotransporter (Nakhoul, N.L., et al. Am J
  • proximal tubule reabsorbs some 85% of the filtered HCO ⁇ 3 and 70% of the filtered Na + .
  • the electrogenic Na + :HCO " 3 cotransporter carries the vast majority of this HCO ⁇ 3 , and about 5% of the Na + .
  • electrogenic Na + :HCO _ 3 cotransporters have subsequently been studied in several epithelial cell lines (Jentsch, T.J., et al. J Membrane Biol (1984) 81 :189-204; Jentsch, T.J., et al. J Biol Chem (1985) 260:15554-15560), intact rat and rabbit proximal tubules (Alpern, R.J.
  • the electrogenicity of the cotransporter implies that each Na + moves with two or more HCO ⁇ 3 or HCO ⁇ 3 equivalents (e.g., COf).
  • HCO ⁇ 3 or HCO ⁇ 3 equivalents e.g., COf.
  • the Na + :HCO ⁇ 3 stoichiometry has not been directly measured. However, it has been deduced by Soleimani, M., et al. J Clin Invest
  • the HCO ⁇ 3 transporters shown in Figure 3 might be thought of as a functional family of transporters that may or may not be related at the molecular genetic level.
  • the renal electrogenic Na + :HCO _ 3 cotransporter, with a Na + :HCO ⁇ 3 stoichiometry of 1 :3, is one of as many as four Na + -coupled HCO ⁇ 3 transporters, as well as a Cr HCO ⁇ 3 exchanger and a K + :HCO ⁇ 3 cotransporter.
  • the Na + -driven C17HCO ⁇ 3 exchanger is the major acid extruder in a wide variety of animal cells.
  • the Na + -driven CF7 ⁇ CO ⁇ 3 exchanger is electroneutral and has an absolute requirement for C ⁇ ⁇ .
  • the Na + -driven CF/ ⁇ CO ⁇ 3 exchanger normally moves
  • the O HC0 ⁇ 3 exchanger sometimes referred to as the Na -independent C17HCO ⁇ 3 exchanger to distinguish it from the Na + -driven C17HCO 3 exchanger, was the first HCO ⁇ 3 transporter to be described.
  • the first of these to be cloned was the band-3 protein of red blood cells, also known as AE-1 (for Anion Exchanger) (Kopito, R.R. et al. Nature (1985) 316:234-238).
  • AE-1 plays a major role in C0 2 carriage by erythrocytes.
  • Ci7HCO ⁇ 3 exchangers Two related Ci7HCO ⁇ 3 exchangers, AE-2 and AE-3, are present in a wide variety of non-erythroid cells. Because they normally mediate the exchange of extracellular Cl ⁇ for intracellular HCO ⁇ 3 , AE-2 and AE-3 function as intracellular acid loaders.
  • the present invention provides isolated nucleic acid molecules that encode various members of the BT superfamily of proteins which are useful in producing the transporter for use in binding/modulation assays and for use in diagnostic screens.
  • the present invention is based, in part, on the isolation and identification of nucleic acid molecules that encode proteins that are members of the 1:3 Na/HCO 3 family of proteins, hereinafter NBC genes or NBC, or NBC encoding nucleic acid molecules (N[a Bicarbonate Cotransporter).
  • NBC genes or NBC or NBC encoding nucleic acid molecules
  • N[a Bicarbonate Cotransporter an NBC encoding nucleic acid molecule was isolated from salamander, the sNBC gene, from rat, the rNBC gene, and from humans, the hNBC gene.
  • the present invention provides isolated nucleic acid molecules that encode an NBC protein, and in particular an NBC proteins from salamanders, rats and humans.
  • Such nucleic acid molecules can be in an isolated form, or can be operably linked to expression-control elements or vector sequences.
  • the present invention further provides methods of identifying nucleic acid molecules that encode other members of the NBC family of proteins. Specifically, nucleic acid sequences that encode the NBC proteins herein described can be used as probes, or to generate PCR primers, in methods that can be used to identify nucleic acid molecules that encode other members of the NBC family of proteins. The present invention further provides methods of identifying other members of the
  • BT Bicarbonate Transporter
  • the herein described nucleic acid sequences that encode the NBC proteins can be used as probes, or to generate PCR primers, in methods to identify nucleic acid molecules that encode other members of the BT superfamily of proteins. These include members of the 1 :2 Na/HC0 3 cotransporter family, the 1 :1 Na/HC0 3 cotransporter family, the K/HC0 3 cotransporter family and the Na driven C1-HC0 3 exchanger family of proteins.
  • the present invention further provides antibodies that bind to the NBC proteins of the present invention.
  • Cloning of several NBC genes, a salamander NBC gene, a rat NBC gene and a human NBC gene and the elucidation of the entire amino acid sequence of several NBC proteins allows one to select epitopes of the NBC protein for generating novel anti-NBC antibodies.
  • Such antibodies can be either polyclonal or monoclonal.
  • Anti-NBC antibodies can be used in a variety of diagnostic formats and for a variety of therapeutic methods.
  • the present invention further provides methods for determining whether binding partners of the NBC proteins exist and isolating the binding partners if present.
  • NBC binding partners are isolated using the NBC protein, or a fragment thereof, as a capture probe.
  • NBC-encoding nucleic acid molecules can be used as bait in the yeast two-hybrid system to screen an expression library and identify genes that encode proteins that bind to an NBC protein. Binding partners isolated by these methods are useful in preparing antibodies and also serve as targets for drug development.
  • the present invention further provides methods to identify agents that can block or modulate the activity of an NBC protein.
  • an agent can be tested for the ability to modulate the activity of an NBC protein by identifying agents that bind to an NBC protein or agents that block, reduce, activate or otherwise modulate the activity of an NBC protein.
  • Such methods are accomplished by contacting NBC, or a fragment thereof, with a test agent, optionally in the presence of a binding partner, and determining whether the test agent binds to the NBC protein and/or blocks or reduces the binding of the NBC protein to the binding partner.
  • Such methods can be used to identify agonist and antagonists of an NBC protein.
  • the present invention further provides methods for reducing or increasing the activity of an NBC protein.
  • agonists of an NBC protein can be used to stimulate the activity of the NBC protein whereas antagonists of an NBC protein can be used to decrease the activity of an NBC protein.
  • Such methods provides means for controlling and/or altering extra- or intracellular pH, Na + concentration and HCO ⁇ 3 concentration, as well as the concentrations of others ions, the transport of which is linked to the transport of Na + (e.g., Ca ⁇ ) or of HCO " 3 (e.g., CO.
  • the present invention further provides methods of regulating the expression of an NBC-encoding nucleic acid molecule within a cell.
  • Expression of an NBC-encoding nucleic acid molecule within a cell can be regulated so as to stimulate or inhibit the production of an NBC protein.
  • Genetic manipulation within an organism can be used to alter the expression of an NBC gene or the production of an NBC protein in an animal model.
  • an NBC gene can be altered to correct a genetic deficiency; peptide modulators of NBC activity can be produced within a target cell using genetic transformation methods to introduce a modulator encoding nucleic acid molecules into a target cell; etc.
  • the use of nucleic acids for antisense and triple helix therapies and intraventions are expressly contemplated.
  • Agonists, antagonists and altering NBC expression can be used as means for treating/modulating biological and pathological processes that require an NBC protein.
  • agonists and methods that reduce or stimulate NBC protein production can be used to alter intracellular or intracellular pH, Na + and/or HCO ⁇ 3 concentration.
  • FIG. 1 Model of HCO ⁇ 3 reabsorption by the mammalian renal proximal tubule.
  • the renal electrogenic Na :HCO ⁇ 3 cotransporter which has a Na + :HCO ⁇ 3 stoichiometry of 1 :3, is the major route for transferring HCO ⁇ 3 from the proximal-tubule cell to the blood and is responsible for moving as much as -90% of the HCO ⁇ 3 transported by the proximal tubule, and -75% of the HCO ⁇ 3 filtered by the glomeruli.
  • the cotransporter is also responsible for directly moving -5% of the Na + from cell to blood. As such, the cotransporter functions as an auxiliary Na + pump.
  • the steady-state pH, of proximal-tubule cells in CO 2 / ⁇ CO ⁇ 3 is -7.1 - -7.5, being more alkaline near the glomerulus.
  • the pH of the luminal fluid falls from 7.4 at the glomerulus to -6.6 at the end of the proximal tubule.
  • the membrane voltage across the basolateral membrane is more negative than -60 mV (Boulpaep, E.L., Kidney Int. (1976) 9, 88-102).
  • the lumen is a few mV negative with respect to the blood.
  • Figure 3 Major HCO ⁇ 3 transporters in animal cells. Three of these transporters normally cause pH, to decrease: the CF/ ⁇ CO ⁇ 3 exchanger, the 1 :3 electrogenic Na + :HCO ⁇ 3 cotransporter, and the K + :HCO ⁇ 3 cotransporter. Three others normally cause pH, to increase: the Na + -driven CF7 ⁇ CO ⁇ 3 exchanger, the 1:2 electrogenic Na + :HCO ⁇ 3 cotransporter and the 1 :1 electroneutral Na + :HCO ⁇ 3 cotransporter.
  • Figure 4 Injecting RNA and assaying it for activity of an electrogenic Na :HCO ⁇ 3 cotransporter.
  • poly(A) + RNA or cRNA synthesized from NBC cDNA
  • aXenopus l ⁇ evis oocytes After several days, the oocyte is impaled with pH and voltage electrodes in order to monitor the pH, and V m changes characteristic of the electrogenic Na + :HCO ⁇ 3 cotransporter.
  • FIG. 5 Amino acid sequence of NBC.
  • a 4,078 bp cDNA encoding the renal electrogenic Na / Bicarbonate Cotransporter (NBC) was isolated by screening a size- selected (3.5-5.0 kb) Ambystom ⁇ -kidney cDNA library for electrogenic cotransport of Na + and HCO3 (see Figure 8 for assay).
  • the cDNA has a poly(A) + tail, an open reading frame from nucleotides 33 to 3107 encoding a 1025 amino acid protein, and a large 3 '-untranslated region (-1 kb, not shown). Rescreening the library led to the identification of an additional
  • a DIDS-binding motif of KL(X)K is present in the AE family (Kopito, R.R., et al., Cell (1989) 59, 927- 937).
  • NBC has the sequence KMIK (558-561), suggesting a K(Y)(X)K motif, where Y is M or L.
  • NBC contains the AE consensus DIDS-binding motif, KLKK, at 768-771.
  • C Dendrogram showing relationships among sNBC, and three most homologous members of the AE family (GenBank Accession #'s: S03074, S21086, A42497). The divergence is indicated by the total length of the line from one sequence to another. Using DNAstar (Lasergene, Madison, WI) NBC is 35%, 33%, and 34%) similar to AE3, AE2, and AE3, respectively.
  • D Membrane model of NBC protein. Putative MSDs are indicated by numbered rectangles. DIDS-binding motifs are indicated as green diamonds. Of 8 consensus N-linked glycosylation sites, only 4 (N-glyc in red) are predicted to be extracellular (amino acids 591, 596, 609, and 617; not 34, 160, 209 and
  • Ser 982 predicted to be intracellular, is the only consensus protein kinase A (PKA, pink triangle) site. Of the 15 consensus sites for protein kinase C (PKC, purple circles), 12 are predicted to be intracellular (Ser 18, Thr 85, Thr 173, Thr 206, Ser 219, Thr 345, Ser 357, Thr 394, Thr 706, Ser 810, Ser 1000, Ser 1020), and 3 to be extracellular (Ser 626, Thr 671, Thr 678).
  • PKA consensus protein kinase A
  • PLC consensus protein kinase C
  • D Membrane model of NBC protein. Because hydropathy plots of NBC and the AEs suggest a similar structure, our NBC model is based on those for the AEs (Reithmeier, R.A.F.
  • PLC protein kinase C
  • 12 are predicted to be intracellular (Ser 18, Thr 85, Thr 173, Thr 206, Ser 219, Thr 345, Ser 357, Thr 394, Thr 706, Ser 810, Ser 1000, Ser 1020), and 3 to be extracellular (Ser 626, Thr 671, Thr 678).
  • FIG. 6 High-stringency northern analysis of poly(A) + RNA from Ambystoma tissues probed with 32p-i aD eled NBC-cDNA, and exposed for 21 h. After a 10-h exposure (not shown), a 4.1 kb transcript was found in the kidney lanes only. After 21 hours of exposure, or longer (not shown), similar transcripts are seen in bladder, small intestine, large intestine, brain and eye.
  • FIG. 7 Expression-cloning assay m Xenopus oocytes.
  • A Control oocyte injected with water. The external solution (pH 7.5) was switched from ND96 solution (C0 2 /HCO ⁇ 3 free) to a solution buffered with 1.5% CO 2 / 10 mM HCO ⁇ 3 . Na + was then removed in the continuous presence of the CO 2 /HCO ⁇ 3 . In water-injected oocytes, removing Na from ND96 (not shown) also elicits a hype ⁇ olarization without changing the pH,.
  • B Oocyte injected with Ambystoma kidney poly(A) + RNA. The C0 2 /HCO ⁇ 3 was added before the portion of the experiment shown. The protocol was the same as in panel A.
  • FIG. 8 Physiology of NBC clone expressed in oocytes. A, DIDS sensitivity. The external solution was switched from ND96 solution (CO 2 /HCO ⁇ 3 free) to 1.5% CO 2 / 10 mM
  • HCO ⁇ 3 at a fixed pH 7.5. Na was then removed four times in the continuous presence of the CO 2 /HCO ⁇ 3 . The last two Na + removals were made in the continuous presence of 200 ⁇ M 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS). In the presence of DIDS, the response to Na removal was similar to that observed in water-injected oocytes in the absence of DIDS ( Figure 7A). B, HCO ⁇ 3 dependence. From a resting value of 7.2 to 7.4 in ND96, pH; decreased to -7.0 upon adding either 1.5% CO 2 / 10 mM HCO " 3 or 10 mM butyrate.
  • DIDS 4,4'-diisothiocyano-2,2'-stilbene disulfonate
  • the oocyte was voltage- clamped at-60 mV as we switched from ND96 to a solution buffered with 1.5% C0 2 / 10 mM HCO _ 3 .
  • the -50 nA outward current elicited by C0 2 /HCO ⁇ 3 corresponds to a -50 mV hype ⁇ olarization observed in undamped cells; the -50 nA inward current elicited by Na removal corresponds to a -50 mV depolarization observed in undamped cells.
  • NBC expression does not significantly change the native oocyte resistance of -1 M ⁇ .
  • Figure 10 Sequence comparison of two partial length rat-brain NBC-related clones and a portion of rkNBC.
  • Figure 11 Sequence comparison of a partial length rat-brain NBC-related clone and a portion of rkNBC.
  • Figure 12 Representation of the NBC cDNA with the location of various DNA oligonucleotide primers.
  • Figure 14 PAC DNA PCR products using human NBC primers to predict regions for introns.
  • FIG. 1 Sequences for human heart NBC (hhNBC).
  • FIG. 1 Nucleotide sequence for rat aorta NBC (raNBC).
  • Figure 18 Nucleotide sequence for NT2-1 A.
  • FIG. 20 Sequences for SI 1.
  • FIG. 21 Western Blot of Crude Membrane Extracts.
  • the antisera was used to probe western blots of crude membranes isolated from Ambystoma tigrinum, rat and rabbit.
  • Figure 22 External HC0 3 dependence of rkNBC expressed in Xenopus oocyte.
  • Figure 23 External HC0 3 dependence of akNBC expressed in Xenopus oocyte.
  • Figure 25 Effect of SO on DIDS sensitive component of HCO 3 -induced pH, increase in Xenopus oocyte expressing rkNBC.
  • Figure 26 Sequences for human kidney NBC (hkNBC, sometimes designated as hkyNBC).
  • Figure 27 Sequences for human pancreas NBC (hpNBC, sometimes called hpanNBC).
  • hpNBC pancreas NBC
  • BT Bicarbonate Transporters
  • Cotransporters had been described. However, due to their size and the transmembrane nature of the protein, little advancement had been made in their characterization and in identifying agonists and antagonists of the cotransporter.
  • the present invention is based in part on cloning of nucleic acid molecules from salamanders, rats and humans that encode proteins that are 1 :3 cotransporters of sodium and bicarbonate, hereinafter the NBC proteins or NBC.
  • the cloning of nucleic acid molecules that encode members of the NBC family of proteins makes possible 1) the isolation and characterization of other NBC- and/or BT-encoding nucleic acid molecules, 2) the characterization of members of the NBC family of proteins, 3) the ability to screen for agonists and antagonist of the NBC protein, 4) the diagnosis of biological and pathological processes that involve normal or aberrant NBC activity, 5) therapeutic methods of controlling/altering NBC activity/expression, 6) the identification of binding partners of the NBC proteins, and 7) generation of animal models in which NBC expression has been altered.
  • the NBC proteins can be used as agents, or serve as targets for agents, that can be used to inhibit or stimulate the activity of the sodium/bicarbonate transporter, for example, to alter extra- or intracellular
  • the present invention provides the ability to produce and isolate NBC proteins, as well as allelic variants of the NBC proteins, and conservative amino acid substitutions of the NBC proteins.
  • an NBC protein refers to a protein that has the amino acid sequence of human, rat or salamander NBC depicted in Figure 5.
  • hNBC will refer specifically to human NBC protein
  • rNBC will refer specifically to rat NBC protein (-1035 amino acids in length)
  • sNBC will refer specifically to salamander NBC protein (-1025 amino acids in length).
  • NBC NBC protein
  • sNBC salamander NBC protein
  • NBC proteins also includes naturally occurring allelic variants of the NBC proteins of the present invention, naturally occurring proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the requisite ability to cotransport 1 :3 sodium and bicarbonate unless the specific allelic variant encodes an NBC protein with altered activity.
  • the NBC family of proteins also includes NBC proteins that have been isolated from organisms in addition to humans, rats and salamanders. The methods used to identify and isolate other members of the NBC family of proteins are described below.
  • NBC is a member of the Bicarbonate Transporter (BT) superfamily of proteins.
  • the superfamily of proteins includes the previously cloned C1-HC0 3 exchanges family (AE1-3), the presently cloned 1:3 Na/HCO 3 cotransporter family (NBC), the 1 :2 Na/HCO 3 cotransporter family, the 1 : 1 Na/HC0 3 cotransporter family, the K/ ⁇ CO 3 cotransporter family and the Na-driven Cl-HCO 3 exchanger.
  • NBC is the first member of the cotransporter (1 :3 or 1 :2) or cation coupled transporter/exchanger families of BT superfamily that has been cloned.
  • NBC amino-acid sequence has little similarity to the amino-acid sequences of the AE family at either the amino or carboxy termini.
  • NBC has a much longer 5,6 exofacial loop than the AE family.
  • NBC has a highly unusual, hydrophilic sequence (“EKDKKKKEDEK-KKKKKK”) near its carboxy terminus, a stretch of 17 amino acids in which each is either positively or negatively charged.
  • NBC differs functionally from the AE family in two important ways.
  • the NBC proteins of the present invention are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the NBC protein from cellular constituents that are normally associated with the NBC protein.
  • a skilled artisan can readily employ standard purification methods to obtain an isolated NBC protein. The nature and degree of isolation will depend on the intended use.
  • the NBC proteins of the present invention further include conservative variants of the NBC proteins herein described. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the ability of the NBC protein to function as a Na/HC0 3 cotransporter.
  • a substitution, insertion or deletion is said to adversely affect the NBC protein when the altered sequence prevents the NBC protein from cotransporting Na/HCO 3 .
  • the overall charge, structure or hydrophobic/hydrophilic properties of NBC can be altered without adversely affecting the activity of NBC.
  • the amino acid sequence of NBC can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the activity of NBC.
  • NBC family of proteins will have the ability to cotransport Na/HCO 3 .
  • Such proteins will ordinarily have an amino acid sequence having at least about 75% amino acid sequence identity with the human NBC sequence, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95%.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and including any conservative substitutions as being homologous.
  • N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
  • fragments of the NBC proteins of the present invention are particularly useful in generating domain-specific antibodies, in identifying agents that bind to an NBC protein and in identifying NBC-binding partners.
  • Fragments of the NBC protein can be generated using standard peptide synthesis technology and the amino-acid sequences disclosed herein. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode fragments of the NBC protein.
  • Figure 5d identifies domains that constitute preferred fragments of the NBC proteins of the present invention.
  • the NBC proteins of the present invention include molecules having the amino acid sequences disclosed in Figure 5; fragments thereof having a consecutive sequence of at least about 3, 5, 10 or 15 amino-acid residues of the NBC protein; amino acid sequence variants of such sequence wherein an amino acid residue has been inserted N- or C-terminal to, or within, the disclosed NBC sequence; amino acid sequence variants of the disclosed NBC sequence, or their fragments as defined above, that have been substituted by another residue.
  • Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding NBC proteins of other animal species, including but not limited to rabbit, rat, murine, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the NBC family of proteins; and derivatives wherein the NBC protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • a detectable moiety such as an enzyme or radioisotope
  • members of the NBC family of proteins can be used for, but are not limited to, 1) a target to identify agents that block or stimulate NBC mediated pH regulation, 2) a target or bait to identify and isolate binding partners that bind NBC,
  • NBC and AE proteins can be used as tools in defining domain structure/activity of the NBC family of proteins. Accordingly, the present invention provides protein chimeras containing a fragment of an NBC protein of the present invention.
  • a chimera is a protein that is made up of segments that are not normally associated with each other.
  • a chimera of an NBC protein can be made up of an NBC protein in which the cytoplasmic C-terminal region is replaced by the C-terminal region of an AE protein.
  • the preferred chimeras will contain one or more intracellular or extracellular domains of an NBC protein.
  • recombinant DNA methods are used to generate a nucleic acid molecule that encodes the chimera.
  • the methods for generating rDNA molecules are described in detail below. A skilled artisan can readily use such procedures to generate chimeric proteins containing one or more fragments of the NBC proteins of the present invention.
  • the present invention further provides antibodies that selectively bind one or more of the NBC proteins of the present invention.
  • Anti-NBC antibodies that are particularly contemplated include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complement determining regions.
  • Antibodies are generally prepared by immunizing a suitable mammalian host using an NBC protein, or fragment, in isolated or immunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). Regions of the NBC proteins herein described that are predicted as being highly antigenic can be readily identified using art known protein modeling methods (for example regions identified in Figure 5B.
  • Figure 5C provides a predicted structure of an NBC protein and identifies extra and intracellular domains.
  • Figure 5A identifies regions that are conserved amongst hNBC, sNBC and rNBC. In the examples, antibodies are described that were generated against fusion proteins consisting of residues
  • a protein for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are well known in the art.
  • a carrier such as BSA, KLH, or other carrier proteins
  • direct conjugation using, for example, carbodiimide reagents may be used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be effective.
  • Administration of the NBC immunogen is conducted generally by injection over a suitable time period and with use of a suitable adjuvant, as is generally understood in the art.
  • titers of antibodies can be taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete a desired monoclonal antibody may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the NBC protein or peptide fragment.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the transporter can also be produced in the context of chimeric or CDR grafted antibodies of multiple species origin.
  • anti-NBC antibodies are useful as modulators of NBC activity, are useful in immunoassays for detecting NBC expression/activity and for purifying an NBC protein.
  • the present invention is based in part on isolating nucleic acid molecules from salamander, rats and humans that encode members of the NBC family of proteins. Accordingly, the present invention further provides nucleic acid molecules that encode an NBC protein as herein defined, preferably in isolated form.
  • all NBC encoding nucleic acid molecules will be referred to as NBC encoding nucleic acid molecules, NBC genes, or NBC.
  • the specifically identified NBC encoding nucleic acid molecules are provided in Figures 5, 9 and 10. Sequences are provided for nucleic acid molecules that encode hNBC (human), rNBC (rat, two isoforms) or sNBC (salamander), the hNBC, rNBC and sNBC genes respectively.
  • nucleic acid molecule is defined as RNA or DNA that encodes a peptide as defined above, or is complementary to a nucleic acid sequence encoding such peptides, or hybridizes to such a nucleic acid and remains stably bound to it under appropriate stringent conditions, or encodes a polypeptide sharing at least 75% sequence identity, preferably at least 80%, and more preferably at least 85%, with the peptide sequences.
  • genomic DNA, cDNA, mRNA and antisense molecules as well as nucleic acids based on an alternative backbone or including alternative bases whether derived from natural sources or synthesized.
  • hybridizing or complementary nucleic acid is defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to a nucleic acid encoding an NBC protein according to the present invention.
  • stringent conditions are conditions in which hybridization yields a clear and detectable sequence. Stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C. Another example is use of 50% formamide, 5 x SSC
  • nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.
  • a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated NBC encoding nucleic acid molecule.
  • the present invention further provides fragments of the NBC encoding nucleic acid molecules of the present invention.
  • a fragment of an NBC-encoding nucleic acid molecule refers to a small portion of the entire protein encoding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the NBC protein, such as the intracellular C-terminal or N- terminal fragments, the fragment will need to be large enough to encode the functional region(s) of the NBC protein. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming.
  • Fragments of the NBC gene that are particularly useful as selective hybridization probes or PCR primers can be readily identified using art know PCR primer selection methods. Such fragments contain regions that are conserved among hNBC, rNBC and sNBC and regions of homology that are shared with the previously identified AE class of proteins.
  • Figure 5B and D identifies potential domains of the NBC proteins.
  • Fragments of the NBC encoding nucleic acid molecules of the present invention i.e., synthetic oligonucleotides
  • PCR polymerase chain reaction
  • Fragments of the NBC encoding nucleic acid molecules of the present invention can easily be synthesized by chemical techniques, for example, the phosphotri ester method of Matteucci, et al, J Am Chem Soc (1981) 103:3185-3191 or using automated synthesis methods.
  • larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the NBC gene, followed by ligation of oligonucleotides to build the complete modified NBC gene.
  • the NBC-encoding nucleic acid molecules of the present invention further include allelic variants of the NBC genes herein disclosed as well as alternative splice forms that are active in tissues other than the kidney. Specifically, the NBC-encoding nucleic acid molecules herein described are expressed in kidney cells. Identical and other forms of the NBC proteins are expressed in tissues such as the brain and the heart. The present invention contemplates such nucleic acid molecules as being within the scope of the invention. A skilled artisan can readily use the methods described below to isolate other forms of the NBC-encoding nucleic acid molecules of the present invention.
  • the NBC-encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe pu ⁇ oses.
  • probes can be used to identify other members of the NBC family of proteins and as described below, such probes can be used to detect NBC expression.
  • labels include, but are not limited to, biotin, radiolabeled nucleotides, biotin, chromaphores and the like. A skilled artisan can employ any of the art known labels to obtain a labeled NBC encoding nucleic acid molecule.
  • a skilled artisan can readily use the amino acid sequence of NBC to generate antibody probes to screen expression libraries prepared from cells.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified NBC protein or a fusion protein generated by recombinant techniques (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as lambda gtll library, to obtain the appropriate coding sequence for NBC, or other members of the NBC family of proteins.
  • the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.
  • Figures 5 B and D identify important antigenic and/or putative operative domains found in the NBC protein sequence. Such regions are preferred sources of antigenic portions of the NBC protein for the production of probe, diagnostic, and therapeutic antibodies.
  • a portion of the NBC encoding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the NBC family of proteins from any mammalian organisms that contains such a protein.
  • Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives. This method was employed to isolate the hNBC- and the rNBC-encoding sequences, using the sNBC encoding sequences as a probe.
  • pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an NBC-encoding nucleic acid molecule.
  • PCR polymerase chain reaction
  • a PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other NBC encoding nucleic acid molecules.
  • conserveed NBC sequences found in Figure 9 are regions of the NBC gene that are particularly well suited for use as a probe or as primers.
  • the NBC encoding nucleic acid molecules of the present invention are particularly well suited for isolating NBC encoding nucleic acid molecules that are expressed in organs such as the brain and heart as well as nucleic acid molecules that encode the related 1 :2 Na/HCO 3 cotransporter family, the 1 : 1 Na/HCO 3 cotransporter family, the K/HCO 3 cotransporter family and the Na-driven Cl- HCO 3 exchanger family.
  • the present invention further provides recombinant DNA molecules (rDNAs) that contain an NBC encoding sequence, or a fragment thereof.
  • a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in vitro. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989).
  • an NBC encoding DNA sequence is operably linked to one or more expression control sequences and/or vector sequences.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the NBC encoding sequences included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, transcription terminators and other regulatory elements.
  • inducible promoters that is readily controlled, such as being responsive to a nutrient in the host cell's medium, is used.
  • the vector containing an NBC encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic host cell such as a bacterial host cell, transformed therewith.
  • vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) of the NBC encoding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ.
  • Expression vectors compatible with eukaryotic cells can also be used to form rDNA molecules that contain an NBC encoding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. Southern et al, J Mol Anal Genet ( 1982) 1:327-341.
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co- transfection of the host cell, and selected by culturing in the presence of the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes an NBC protein of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of an NBC protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the NBC gene product.
  • Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line, the most preferred being cells that do not naturally express an NBC protein.
  • Any prokaryotic host can be used to express an NBC-encoding rDNA molecule.
  • the preferred prokaryotic host is E. coli.
  • Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al. , Proc Acad Sci USA ( 1972) 69 :2110; and Maniatis et al. , Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • electroporation, cationic lipid or salt treatment methods are typically employed, see, for example, Graham et al, Virol (1973) 52:456; Wigler et al, Proc Natl Acad Sci USA (1979) 76:1373-76.
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J Mol Biol (1975) 98:503, or Berent et al, Biotech (1985) 3:208 or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing an NBC protein that uses one of the NBC encoding nucleic acid molecules herein described.
  • the production of a recombinant form of an NBC protein typically involves the following steps.
  • nucleic acid molecule that encodes an NBC protein, such as the nucleic acid molecule depicted in Figure 5. If the NBC encoding sequence is uninterrupted by introns, as are the sequences provided in Figure 5 and 9, it is directly suitable for expression in any host. If not, then a spliced form of the NBC encoding nucleic acid molecule can be generated and used or the infron-containing nucleic acid molecule can be used in a compatible eukaryotic expression system.
  • the NBC-encoding nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the NBC encoding sequences.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the NBC protein.
  • the NBC protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with NBC encoding sequences to produce an NBC protein.
  • Particularly well suited are expression system that result in the production of lipid vesicles containing the expressed protein.
  • Such lipid containing vesicles are well suited for identifying agonists and antagonists of the NBC protein.
  • NBC is a member of the BT superfamily of proteins and is involved in the cotransport of sodium and bicarbonate. This cotransport is used by cells and tissues as a means of altering extra- or intracellular pH. Accordingly, NBC can be used in methods to alter the extra- or intracellular pH or concentration of Na + or HCO ⁇ 3 . In general, cellular pH can be altered by altering the expression of an NBC protein or the activity of an NBC protein.
  • an NBC protein or NBC gene expression can be used as a target for means to alter extra- or intracellular pH, or sodium or bicarbonate concentrations.
  • an NBC gene can be introduced and expressed in cells to increase NBC expression.
  • This provides a means and methods for altering extra- and intracellular pH.
  • pathological conditions characterized by inappropriate extra- or intracellular pH. For example, water retention, increase blood pressure, chronic respiratory and metabolic acidosis, inflammation, cell proliferation (as in cancer), sperm activation inactivation, hydroencephaly, epilepsy, altered breathing rate, glaucoma, colitis, are all associated with abnormal intracellular and/or extracellular pH.
  • NBC activity/expression is targeted as a means of treating these conditions.
  • Various methods for regulating NBC activity/expression are discussed in detail below.
  • Another embodiment of the present invention provides methods for identifying agents that are agonists or antagonists of the NBC proteins herein described. Specifically, agonist and antagonist of an NBC protein can be first identified by the ability of the agent to bind to an NBC protein. Agents that bind to an NBC protein can then be tested for the ability to stimulate or block Na HC0 3 cotransport in an NBC-expressing cell.
  • an ⁇ BC protein is mixed with an agent. After mixing under conditions that allow association of ⁇ BC with the agent, the mixture is analyzed to determine if the agent bound the ⁇ BC protein. Agonists and antagonists are identified as being able to bind to an ⁇ BC protein.
  • the ⁇ BC protein used in the above assay can either be an isolated and fully characterized protein, can be a partially purified protein, can be a cell that has been altered to express an ⁇ BC protein or can be a fraction of a cell that has been altered to express an ⁇ BC protein. Further, the ⁇ BC protein can be the entire ⁇ BC protein or a specific fragment of the ⁇ BC protein. Particularly useful fragments include, but are not limited to the intracellular and extracellular domain and the DIDS binding domain. It will be apparent to one of ordinary skill in the art that so long as the ⁇ BC protein can be assayed for agent binding, e.g., by a shift in molecular weight or change in cellular pH, the present assay can be used.
  • the method used to identify whether an agent binds to an ⁇ BC protein will be based primarily of the nature of the ⁇ BC protein used. For example, a gel retardation assay can be used to determine whether an agent binds to a soluble fragment of an ⁇ BC protein whereas patch clamping, voltage clamping, pH-sensitive microprobes or pH-sensitive chromaphores can be used to determine whether an agent binds to a cell expressing an ⁇ BC protein. A skilled artisan can readily employ numerous techniques for determining whether a particular agent binds to an NBC protein.
  • the agent can be further tested for the ability to modulate NBC activity using a cell or oocyte expression system and an assay that detects NBC activity.
  • a cell or oocyte expression system and an assay that detects NBC activity.
  • voltage or patch clamping, pH-sensitive microprobes or pH- sensitive chromaphores and expression in Xenopus oocytes or recombinant host cells can be used to determine whether an agent that binds an NBC protein can agonize or antagonize NBC activity.
  • an agent is said to antagonize NBC activity when the agent reduces NBC activity.
  • the preferred antagonist will selectively antagonize NBC, not affecting any other cellular proteins. Further, the preferred antagonist will reduce NBC activity by more than 50%, more preferably by more than 90%, most preferably eliminating all NBC activity.
  • an agent is said to agonize NBC activity when the agent increases NBC activity.
  • the preferred agonist will selectively agonize NBC, not affecting any other cellular proteins. Further, the preferred agonist will increase NBC activity by more than 50%), more preferably by more than 90%, most preferably more than doubling the level of NBC activity.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences of the NBC protein.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the NBC protein.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to a fragment of an NBC protein.
  • agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • One class of agents of the present invention are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of the NBC protein.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of the NBC protein. As described above, antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the NBC protein intended to be targeted by the antibodies. Critical regions include the domains identified in Figures 5B and 5D.
  • NBC is involved in regulating intracellular and extracellular pH, as well as Na + and HCO ⁇ 3 concentrations.
  • Agents that bind an NBC protein and act as an agonist or antagonist of an NBC protein can be used to modulate biological and pathologic processes associated with NBC function and activity.
  • a biological or pathological process mediated by NBC can be modulated by administering to a subject an agent that binds to an NBC protein and acts as an agonist or antagonist of NBC activity.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by NBC.
  • the term "mammal” is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.
  • a biological or pathological process mediated by NBC refers to the wide variety of cellular events mediated by an NBC protein.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • pathological processes mediated by NBC include water retention, increased blood pressure, chronic respiratory and metabolic acidosis, inflammation, cell proliferation (e.g., cancer), sperm activation/inactivation, hydroencephaly, epilepsy, glaucoma and colitis. These pathological process can be modulated using agents that reduce or increase the activity of an NBC protein.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • an agent is said to modulate acidosis when the agent contributes to normal blood pH.
  • Agonists and antagonists of the NBC protein can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. For example, to treat water retention, increased blood pressure, chronic respiratory and metabolic acidosis, inflammation, cancer, sperm activation/inactivation, hydroencephaly, epilepsy, glaucoma, colitis, an agent that modulates NBC activity is administered systemically or locally to the individual being treated. As described below, there are many methods that can readily be adapted to administer such agents.
  • the present invention further provides compositions containing an antagonist or agonist of an NBC protein that is identified by the methods herein described. While individual needs vary, a determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 Tg/kg body wt. The preferred dosages comprise 0.1 to 10 Tg/kg body wt. The most preferred dosages comprise 0.1 to 1 Tg/kg body wt.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dintran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the agents of the present invention that modulate NBC activity can be provided alone, or in combination with another agents that modulate a particular biological or pathological process.
  • an agent of the present invention that reduces NBC activity can be administered in combination with other agents that effect sodium/bicarbonate cotransport.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • NBC proteins Because of the nature of NBC proteins, it is presently unknown whether other cellular constituents, such as signaling proteins or non-protein cofactors, are associated with
  • NBC proteins The cloning of the NBC encoding nucleic acid molecules herein described allows for the detection of such binding partners and their isolation, if they exist Accordingly, another embodiment of the present invention provides methods that can be used to isolate and identify binding partners of NBC.
  • the NBC protein, or a fragment thereof can be used as a capture probe to identify NBC binding partners.
  • an NBC binding partner is a biomolecule (such as a protein, DNA or other cofactor) that binds to NBC and mediates NBC activity.
  • an NBC protein or a fragment of an NBC protein, is mixed with an extract or fraction of a cell that expresses NBC under conditions that allow the association of a binding partner with NBC. After mixing, peptides that have become associated with NBC are separated from the mixture. The binding partner that bound NBC can then be removed and further analyzed.
  • the entire NBC protein can be used.
  • a fragment of an NBC protein can be used. Because the C-terminal and N- terminal of the NBC proteins are large, intracellular regions, a fragment containing either of these regions is particularly well suited for isolating NBC binding partners because such regions are likely to be the contact site of a binding partner if one exists.
  • the other intracellular and extracellular domains identified in Figure 5C can be used.
  • a cellular extract refers to a preparation or fraction that is made from a lysed or disrupted cell.
  • the preferred source of cellular extracts will be cells that naturally express NBC. Examples of such cells include, but are not limited to the kidney, brain, heart, stomach, small and large intestines, pancreas, liver, testis, prostate, sweat and salivary glands, mammary glands, choroid plexus, ciliary body, bone and blood vessels.
  • the extract is mixed with the NBC protein under conditions in which association of NBC with the binding partner can occur.
  • conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of an NBC-expressing cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the NBC with the binding partner.
  • NBC is separated from the mixture.
  • a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to NBC can be used to immunoprecipitate the NBC and associated binding partner. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.
  • the binding partner can be dissociated from the NBC protein using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the NBC protein can be immobilized on a solid support.
  • NBC can be attached to a nitrocellulose matrix or acrylic beads. Attachment of NBC to a solid support further aids in separating a peptide/binding partner pair from other constituents found in the extract.
  • an NBC-encoding nucleic acid molecule can be used in a yeast two- hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the NBC encoding molecules herein described.
  • the NBC binding partners obtained using the above described methods can be used for a variety of pu ⁇ oses.
  • the binding partners can be used to generate antibodies that bind to the NBC binding partner using techniques known in the art.
  • Antibodies that bind an NBC binding partner can be used to assay NBC activity, as a therapeutic agent to modulate a biological or pathological process mediated by NBC, or to purify the binding partner. These uses are described in detail below.
  • the identified binding partner can be use to identify agents that reduce or block the association of NBC with an NBC binding partner.
  • NBC is mixed with an NBC binding partner in the presence and absence of an agent to be tested. After mixing under conditions that allow association of NBC with the NBC binding partner, the two mixtures are analyzed and compared to determine if the agent reduced or blocked the association of NBC with the NBC binding partner. Agents that block or reduce the association of NBC with the NBC binding partner will be identified as decreasing the amount of association present in the sample containing the tested agent.
  • an agent is said to reduce or block NBC/NBC binding partner association when the presence of the agent decreases the extent to which or prevents the
  • NBC binding partner from becoming associated with an NBC protein.
  • One class of agents will reduce or block the association by binding to the NBC binding partner while another class of agents will reduce or block the association by binding to the NBC protein.
  • NBC binding partner that has been identified as being present in a cellular extract. It will be apparent to one of ordinary skill in the art that so long as the NBC binding partner has been characterized by an identifiable property, e.g., molecular weight, the present assay can be used.
  • Agents that block NBC/signaling partner interaction can be agonists or antagonists of NBC activity. Such agonists and antagonists can be used essentially as described above for agents that bind to an NBC protein.
  • the present invention further provides methods for identifying cells expressing active and aberrant forms of an NBC protein as well as techniques that can be applied to diagnose biological and pathological processes associated with NBC activity, the progression of such conditions, the susceptibility of such conditions to treatment and the effectiveness of treatment for such conditions.
  • NBC activity can be identified by determining whether the NBC protein is expressed in a cell. Departure from normal expression, or the expression of an aberrant form, can be used as a means for diagnosing pathological conditions mediated by abnormal NBC activity/expression.
  • a variety of immunological and molecular genetic techniques can be used to determine if an NBC protein, or an NBC encoding mRNA, is produced in a particular cell.
  • an extract of cells is prepared. The extract is then assayed to determine whether NBC is expressed in the cell. The degree of expression provides a measurement of the degree of NBC activity. An increase of expression over normal levels indicates an over- activity of NBC.
  • the measurement of NBC expression can be used as a marker for a variety of pu ⁇ oses. For example in identifying susceptibility or the presence of NBC mediated water retention, increased blood pressure, chronic respiratory and metabolic acidosis, inflammation, cancer, sperm activation/inactivation, hydroencephaly, epilepsy, glaucoma or colitis.
  • NBC expression can also be used in methods to identify agents that increase the expression of a naturally occurring NBC gene.
  • nucleic acid probes that detect mRNA can be used to detect cells or tissues that express an NBC protein. Such cells or tissues can be contacted with a test agent to determine the effects of the agent on NBC expression, agents that activate NBC expression can be used as an agonist of NBC activity whereas agents that decrease NBC activity can be used as an antagonist of NBC activity.
  • the present invention further provides additional methods that can be used to control NBC expression in a cell.
  • the expression of an NBC antisense nucleic acid molecule in a cell can be used as a means of reducing NBC activity.
  • a skilled artisan can readily employ antisense technology with the NBC encoding nucleic acid molecules of the present invention.
  • NBC activity can also be altered by altering the level of expression of naturally occurring NBC genes using agents identified by the methods described above. Agents that increase or decrease the level of NBC expression can be used to regulate NBC activity within cells or tissues.
  • the NBC gene and the NBC protein can also serve as targets for gene therapy in a variety of contexts.
  • NBC-deficient non-human animals can be generated using standard knock-out procedures to inactivate an NBC gene or, if such animals are non- viable, inducible NBC antisense molecules can be used to regulate NBC activity/expression.
  • an animal can be altered so as to contain an NBC or antisense-NBC expression unit that directs the expression of NBC or the antisense molecule in a tissue specific fashion.
  • a non-human mammal for example a mouse or a rat, is generated in which the expression of the NBC gene is altered by inactivation or activation.
  • the NBC-deficient animal the animal that expresses NBC in a tissue specific manner, or an animal that expresses an antisense molecule can be used to 1) identify biological and pathological processes mediated by NBC, 2) identify proteins and other genes that interact with NBC, 3) identify agents that can be exogenously supplied to overcome NBC deficiency and 4) serve as an appropriate screen for identifying mutations within NBC that increase or decrease activity.
  • genetic therapy can be used as a means for modulating an NBC-mediated biological or pathological processes.
  • a genetic expression unit that encodes a modulator of NBC expression, such as an antisense encoding nucleic acid molecule or an NBC encoding nucleic acid molecule.
  • a modulator of NBC expression such as an antisense encoding nucleic acid molecule or an NBC encoding nucleic acid molecule.
  • Such a modulators can either be constitutively produced or inducible within a cell or specific target cell. This allows a continual or inducible supply of a modulator of NBC expression within a subject.
  • RNA was isolated with T ⁇ zol ® reagent (GibcoBRL) from tissues obtained from 25 Ambystoma tignnum, followed by ohgo-dT selection with ohgo-dT cellulose (GibcoBRL) or FastTracks (Ambion) 2 ⁇ g of tissue poly(A) + RNA was electrophoresed m a denatu ⁇ ng (50% formaldehyde) MOPS buffer, and then blotted and UN cross-linked to a Zeta-Probe ® nylon membrane (BioRad) The blot was probed with 32p. ra ndom-hexamer-p ⁇ med D ⁇ A (GibcoBRL) from the open reading frame of NBC using ExpressHyb (Clontech)
  • Electrodes were connected to a high-impedance electrometer and output data acquired by computer. pH electrodes had slopes of -54 to -57 mV / pH unit between pH 6.0 and 8.0
  • the CO 2 /HCO ⁇ 3 -free ⁇ D96 contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1 8 mM CaCl 2 , and 5 mM HEPES (pH 7.5).
  • 10 mM NaHCO 3 replaced 10 mM NaCl Solutions contained 100 ⁇ M ouabain to block the oocyte' s native Na-K pump.
  • cho ne replaced Na +
  • the expression-cloning strategy used was unique both in the assay and the source of the poly(A) RNA.
  • both voltage- and ion-sensitive microelectrodes were used to monitor simultaneously V and pH, of Xenopus laevis oocytes.
  • the protocol was to equilibrate the oocyte with CO 2 /HCO 3 and then remove extracellular Na + . Because the cotransporter exports more HCO ⁇ 3 than Na + , this maneuver leads to a positive-shift in V m and a fall in pH,.
  • poly(A) + RNA from rabbit kidney cortex, the expected V m and pH, signals were not detected.
  • a feature of cells injected with NBC vs. poly(A) + RNA is the immediate hype ⁇ olarization caused by applying C0 2 / ⁇ CO ⁇ 3 ( Figure 8A), due to the inward (i.e., reverse) flux of Na and HCO 3 .
  • 200 ⁇ M DIDS blocks these V m and pH j changes.
  • NBC is Na + dependent and blocked by DIDS.
  • NBC's HCO " 3 dependence was tested.
  • the NBC clone was used to isolate other members of the family and super family of proteins.
  • An isotype variant has been identified in brain ( Figure 10).
  • the brain isoform has an open reading frame that is identical to that of the renal NBC except that, near the C terminus, there is a 98 nucleotide deletion that introduces a frame shift.
  • the deduced amino-acid sequence of the brain isoform is different from that of the renal NBC for the 46 C-terminal amino acids, and is also 15 amino acids longer than the original renal clone.
  • the brain clone differs from the kidney clone over its last 61 amino acids.
  • the brain has the original renal form of NBC. Only the brain, so far, has the unique isoform. Antibodies specific for each of the different isoforms can be readily generated and can be used to distinguish between the original and brain isoform.
  • the antisera was used to probe western blots of crude membranes isolated from Ambystoma tigrinum, rat and rabbit ( Figure 21).
  • the disclosed NBC clone encodes the renal electrogenic Na/HC0 3 cotransporter that mediates HCO 3 efflux and presumably has a Na + :HCO ⁇ 3 stoichiometry of 1 :3.
  • a functionally related cotransporter which mediates both HCO ⁇ 3 influx and efflux and has a stoichiometry of 1 :3, also has been identified in retinal M ⁇ ller cells (Newman, E.A. and Astion, M.L. Glia (1991) 4, 424-428; Newman, E.A. JNeurosci (1996) 16, 159-168).
  • Other cotransporters presumably related to NBC at the molecular level, mediate HCO ⁇ 3 influx.
  • Cotransporters with Na + :HCO ⁇ 3 stoichiometries of 1 :2 are found in glia (Dart, C. and Vaughan- Jones, R.D. J. Physiol. (1992) 451, 365-385), liver (Fitz, J.G., Persico, M., and Scharschmidt, B.F. Am. J. Physiol. (1989) 256, G491-G500; Gleeson, D., Smith, N.D., and Boyer, J.L. J. Clin. Invest (1989) 84, 312-321; Weintraub, W.H. and Machen, T.E. Am. J. Physiol.
  • rat-brain NBC or rbNBC
  • rkNBC rat-brain NBC
  • rkNBC and rbNBC are both present in mRNA from total brain (TB) of rat.
  • TB total brain
  • TB total brain
  • sense primer 5'-ggCgTgTTCTTgTATATgggggTggCCTCA-3'
  • antisense primer 5'- gTTCCTCCAAAgATAATCgTTgTCT-3'.
  • hgPAC human genomic plasmid artificial chromosome
  • hgPAC's were obtained from two different sources: Genome Systems (St. Louis, MO) and Dr. Richard Lifton's laboratory (HHMI and Boyer Center for Molecular Medicine, Yale Univ., School of Medicine). Both library screenings used the same PCR-generated cDNA probe. This PCR- cDNA probe (-1.6 kb) was generated by amplification of the original human kidney clone (h912b) using SSSS_f / DPGE_r.
  • h912b was obtained by screening a human kidney cortex lgtlO library with the EcoRI-fragment of the salamander NBC clone (aNBC-Ambystoma NBC). This EcoRI-fragment is a 3kb fragment which encompasses most (>95%) of the aNBC open reading frame (ORF) or amino acid coding region.
  • ORF open reading frame
  • Blots were washed 3x with 2xSSC / 0.05% SDS at room temperature over 40 min. A final high stringency washes contained O.lxSSC / 0.1 % SDS and was performed at 65 °C over 30-40 min. Blots were wrapped in saran wrap and exposed to autoradiography film at -80 °C for 12-36 hours and then developed.
  • h912b is a 5' human kidney cDNA clone, which was then amplified using PCR between SSSS_f / DPGE_r. This human 5' probe was then random hexamer primed with P-dCTP and run over a G-50 Sephedex column to remove free nucleotides and increase the specific activity of the probe. This 5' probe at high stringency hybridized to all hgPACs except 478, 479, and 994. Even with longer exposure time no reactivity was evident. Banding patterns for the other clones might be suggestive of more than one gene, but the data was not conclusive.
  • Imfr 3' probe
  • imfr is a probe previously discussed in the earlier iteration of this application. It is obtained from a cDNA (human melanocyte origin) found in the I.M.A.G.E consortium dbEST (EST database). This clone begin with "FLDDVIPEKDKKKK" and continues - 500 bp into the untranslated region of the cDNA. Thus, "imfr” does NOT overlap with h912b, though both contain regions of the NBC cDNA(s). Again the probe was generated and purified as above, and the same Southern blots hybridized at high stringency. Only 864 was recognized by this 3' probe. Thus, only hgPAC 864 contains both the 5' and 3' ends of the NBC cDNA by Southern analysis and is therefore, presumed to contain a complete NBC gene.
  • FISH analysis FISH localization carried out on inte ⁇ hase chromosome squashes of human neutropils revealed that there was indeed more than one NBC gene. As indicated above, the hgPACs fall onto 3 human chromosomes: 1, 4, and 17. 994 on chr 1 does not seem to be a true NBC related gene; however, since the hgPAC has not been sequenced it is still formally possible that 994 represents a somewhat distantly related NBC gene.
  • MSTE appears to PRECEDE MEDE in the genomic sequence.
  • clones 929, 952, and 994 represent a separate NBC gene
  • clones 339, 915, and 947 are most likely distinct from the NBC gene which contain both the MSTE .... Intron.... MEDE... start. hgPAC 479.
  • MEDE start has been confirmed by sequencing. Results Genomic DNA Sequence information. To identify the genomic DNA sequences for the human form(s) of NBC, Genome Systems, Inc. used a fragment of the NBC clone IMFR
  • FIG. 9 to screen a PAC (Plasmid Artificial Chromosome) DNA library.
  • This screening identified 5 genomic PAC clones with homology to the human NBC.
  • Five PAC DNA's (15256, 15257, 15258, 15478 & 15479) were identified and obtained from Genome System Inc. Additionally, Dr. Richard P. Lifton (Yale University) also screened his PAC DNA library and identified 7 additional PAC clones (839, 864, 915, 929, 947, 952 and 994).
  • FIGs 12, 13 and 14 Representation of the NBC cDNA with location of various DNA oligonucleotide primers is shown in Figure 12. Each primer is represented by a horizontal bar and is identified by the single letter amino acid code for the first 4 amino acids encoded by DNA oligonucleotide. The sequence of the PCR primers is provided in Figure 13. Using these primers to date we can determine the location of potential introns. Each primer pair will yield a DNA fragment of predicted size (from the nucleotide sequence of NBC cDNA). The yield of a DNA product of a size greater than that of the expected value is an indication of the area of an intron. The following is a description of the areas we have identified for putative introns.
  • Cloning hhNBC The open reading frame of rkNBC was cut into three pieces with BstX I, radiolabeled with a-[ 32 P]-dCTP by random priming, pooled and used to screen a human heart ⁇ ZAP II cDNA library. The titrated plaques (0.64 x 10 6 ) were plated and blotted on nitrocellulose filters. Hybridization was performed in 0.5 M sodium phosphate, 7%>SDS, 1% Bovine Serum Albumin at 65°C overnight. The membranes were washed at 50°C in 1 x SSC/0.1% SDS, and then autoradiographed.
  • the above approach yielded nucleotide sequence data from both the 5' end (clones 15.1, and 1.3) and the 3' end (clones 4.1 and 8.1) of the putative clone. However, we were missing the middle of the putative clone.
  • the upstream primer sequence was 5'-CCG GAG AAG GAC CAG CTG AAG-3', corresponding to a region near the 3' end of clone 15.1, which contains the 5' fragment of hhNBC.
  • the downstream primer sequence was 5'-ATC AGA GTA GGG AGG AAA GAG-3', corresponding to a sequence at the TAG stop codon of clones 4.1.
  • the result of the PCR was a DNA fragment that represented the middle and 3' end of the putative clone, which we sequenced.
  • this sequence is present in the cDNA clone obtained from ⁇ library, it should not be a cloning artifact generated by mis-inco ⁇ oration during PCR.
  • These clones also contained the 3' UTR.
  • the other two clones contained a partial fragment (clone 15.1 and 1.3) corresponding to the N-terminus of hkNBC.
  • the first 85 amino acids of these clones replace the first 41 amino acid residues of hkNBC, with the ATG translation initiation codon in a perfect Kozak sequence.
  • hhNBC Sequence analysis of hhNBC.
  • the open-reading frame of hhNBC encodes 1083 amino acids, with a predicted molecular weight of 121 kDa ( Figure 15).
  • a detailed analysis of the deduced amino acid sequence shows that the unique N-terminus of hhNBC consists of mainly charged amino acids such as aspartate, glutamate, histidine, lysine, or arginine ( Figure 16). These charged residues comprise -50% of the unique 85 amino acids in hhNBC, higher than -22% in the corresponding N-terminal region (41 amino acid residues) of renal NBCs.
  • the hydropathy plot shows that the most N-terminal region of hhNBC is very hydrophilic.
  • hhNBC has the predicted membrane-spanning domains (MSDs) virtually identical to hkNBC and 98% identical to rat NBC homolog (rKNBC), this suggests that the hhNBC has a strong polar or charged amino terminus in the cytoplasmic domain.
  • MSDs membrane-spanning domains
  • rKNBC rat NBC homolog
  • Electrogenic renal NBC and the anion exchangers (AEs) belong to one superfamily of
  • HCO _ 3 transporter Among AEs, hhNBC is structurally closer to AE2 or AE3 than AE1. The N-terminal region of AE2 or AE3 is larger and contains the highly charged amino acids, whereas AE1 does not.
  • AARGGIKCIGGITWYCAYCTIGAY co ⁇ esponding to 2312-2336 of rkNBC sequence and the down stream primer is ICCIGWIACRTAYTGYTCRTAIAC, corresponding to 2581-2605 of rkNBC.
  • inosine base was used for not concerned position.
  • PCR was performed in first three cycles with denaturation at 94°C for 30 sec, annealing at 42°C for 1 min., extension at 72°C for 2 min., and the next 20 cycles with denaturation at 94°C for 30 sec, annealing at 50°C for 1 min., extension at 72°C for 2 min.
  • the PCR product was then diluted to 200 fold with water and the second round PCR was done with the same upstream primer used for the first PCR and the nested downstream primer.
  • the nested primer sequence is IGAIGIIAYICCCATRTADAGRAA, corresponding to 2715-2738 of rkNBC.
  • the PCR product were cloned into the pCRII vector (Invitrogen). The individual PCR products obtained were sequenced at the Kech Sequencing Center at Yale University. Results
  • raNBC The open reading frame of raNBC is 3,783 bp, encoding 1261 amino acids.
  • the raNBC reveals -55% homology to other NBCs and 40-45% to AE2 .
  • a detailed analysis of the deduced amino acid sequence shows that, in contrast to rkNBC, the N- terminus of raNBC is fairly long and consists of many charged amino
  • the hydropathy plot of raNBC is similar to that of rkNBC, with 10 membrane-spanning domains (MSDs) (data not shown).
  • MSDs membrane-spanning domains
  • One putative consensus PKA sites is found at 227.
  • At least three potential N- linked glycosylation site is found at amino acid position 767-770, 777-780, and 787-790.
  • Cloning NT2 The GenbankTM nonredundant EST data base was queried against the raNBC. Two human EST clones (clone ID: 649838, 664517) with -70% homology to raNBC had a highest match score with the smallest probability to be a nonspecific homology. These clones were purchased from the Genome Systems. They were sequenced at the Kech Sequence Center at Yale. The EST clone 649838 contained a 2.5 kb insert fragment consisting of more than half of the 5' coding region of NT2. The second EST clone 664517 contained a -4 kb insert fragment corresponding the more than half of the 3' coding region of NT2. Two clones were ligated at the BamHI site present in the overlapping site.
  • EST clone ID: 649838 contained a 2.5 kb insert fragment consisting of more than half of the 5' coding region of NT2. It has a translation start codon with the stop signal at the upstream region.
  • the second EST clone (clone ID: 664517) contained a -4 kb insert fragment corresponding the more than half of the 3' coding region of NT2. This clone has a translation stop codon and the 3' untranslated region.
  • NT2 clone Two clones overlapped for -1.5 kb, representing that they together encode a full-length cDNA for NT2. We therefore ligated them at the overlapping site.
  • the open reading frame of NT2 clone is approximately 3000 kb, encoding approximately 1000 amino acids (see Figure 18 for NT2-1A and Figure 19 for NT2-2B).
  • raNBC reveals -55% homology to other NBCs and ⁇ 40%> to AEs.
  • a detailed analysis of the deduced amino acid sequence shows that, in contrast to raNBC, both amino and carboxyl terminus of raNBC is fairly short. Some regions are specially absent from
  • NT2 The hydropathy plot of NT2 is similar to that of raNBC (data not shown). There are at least 10 membrane-spanning domains (MSDs). Two putative consensus PKA sites are found at amino acid number 132-135. Four potential N-linked glycosylation site are found at amino acid number 562-565, 572-575, 567-580, 582-585.
  • MSDs membrane-spanning domains
  • Two putative consensus PKA sites are found at amino acid number 132-135.
  • Four potential N-linked glycosylation site are found at amino acid number 562-565, 572-575, 567-580, 582-585.
  • Example 7 Isolation and Sequencing of Sll. Materials and Methods
  • cDNA was made from human polyA RNA with a Sl l cDNA-specific primer LSSD-R (342-369) After tailing the 5' end with CTP, PCR was done with the 5' anchor primer and the downstream gene-specific primer HANS-R (289-321). The PCR product was then diluted to 20 fold with water, and used for the second PCR with the 5 ' AUAP primer and the downstream gene- specific primer EHVP-R (72-99).
  • PCR was done in 30 cycles with denaturation at 94°C for 1 min., annealing at 60°C for 2 min., and extension at 68°C for 3 min.
  • the PCR products was diluted to 40 fold with water, and used for the second nested PCR with the primer CQEM-F and the same vector primers.
  • the resulting PCR products were subcloned into pCRII (Invitrogen) vector and sequenced at the Keck Sequence Center at Yale.
  • Sequencing the fragment from the other end shows no reasonable open reading frames, indicating that the -2 kb fragment should correspond to the most 3' end of Sl l clone.
  • the middle part of the fragment is sequenced using sequencing methods well known to one skilled in the art.
  • Sl l has a 52.4% homology to Ambistoma kidney NBC, and -70% homology to raNBC or NT2, at the nucleotide level.
  • Example 8 Polyclonal Antibodies to rbNBC and rkNBC. Materials and Methods
  • Immunogens were prepared using the maltose-binding protein (MBP) fusion protein system.
  • MBP maltose-binding protein
  • a purified PCR product encoding for either the C- terminal 46 amino acids (a.a.) of rat-kidney NBC (rkNBC), or the C-terminal 61 a.a. of rat- brain NBC (rbNBC) was ligated into the expression vector pMAL-c2 at the EcoRI restriction site.
  • the PCR primers used to obtain the C-terminal 46 a.a. of rkNBC were: 5'- CggAATTCTCTgACTgCCCATACTCAg-3' (sense) and
  • Tissues were isolated from Sprague-Dawley rats, placed in ice- cold homogenization buffer (containing 1 tablet of protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN)/ 10 mis), and homogenized using a polytron. The homogenate was then centrifuged for 15 min. at -1000 g (4°C) to remove cell debris and nuclei. The supernatant was again centrifuged for 45 min at -27,000 g (4°C) to pellet microsomes containing plasma and organellar membranes. The pellet was resuspended in homogenization buffer, and the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit.
  • BCA bicinchoninic acid
  • rbNBC is predominantly expressed in brain.
  • Example 9 Polyclonal Antibodies to NBC-3 and NBC-5 of rkNBC.
  • the respective cDNA sequence was amplified by PCR using modified primers that added an EcoRI restriction site on the 5 ' end, and a stop codon plus an Xbal restriction site on the 3' end (sense primer: 5'-Cg gAA TTC gCg ATT ATT TTT CCA gTC ATg ATC-3 '; antisense primer: 5 '-Cg TCT AgA TCA gCA TgA TgT gTg gCg TTC AAg g-3 ' ).
  • the purified PCR product was digested with EcoRI and Xbal, repurified, and ligated into the MBP expression vector pMAL-c2 (N ⁇ B, Beverly, MA) that had been linearized with Ec ⁇ RUXb ⁇ l.
  • the resulting construct (“pMAL-NBC-5") was transfected into E. coli (DH5 ⁇ , GIBCO BRL, Gaithersburg, MD) and propagated in Luria-Bertani medium containing 100 ⁇ g/ml ampicillin (“LB/Amp").
  • LB/Amp Luria-Bertani medium containing 100 ⁇ g/ml ampicillin
  • Bidirectional sequencing of the purified plasmid confirmed the absence of frameshifts or mutations.
  • we also generated a fusion protein containing amino acids (a.a.) 338-391 of rNBC (“MBP-NBC-3").
  • a third fusion protein contained the ⁇ -fragment of ⁇ -galactosidase ("MBP- ⁇ gal”; transcribed from wildtype pMAL-c2).
  • a fourth fusion protein (“MBP-NH ⁇ 3") contained the C-terminal 131 amino acids of rabbit NHE3 (Biemesderfer et al, 1997).
  • E. coli harboring pMAL-NBC-5 were grown at 37 C in 500 ml LB/Amp. When the culture medium had reached an optical density of 0.4 - 0.6 at 550 nm, expression of the fusion protein gene was initiated by adding isopropylthiogalactoside (IPTG; final concentration 0.3 mM) and allowed to proceed for 4 h. Cells were then pelleted by centrifugation (3,000 g for 15 min at 4 C) and resuspended in 25 ml "column buffer” (in mM: 50 TRIS, 300 NaCl, 1 phenylmethylsulfonyl fluoride (PMSF), 1 Na-EDTA, pH 7.4 with HCI).
  • IPTG isopropylthiogalactoside
  • PMSF phenylmethylsulfonyl fluoride
  • the cell suspension was sonicated vigorously for 20 min on ice.
  • the sonicate was cleared from insoluble matter by centrifugation (15,000 ⁇ m in SS-34, 30 min at 4 C) and diluted with 4 volumes of column buffer.
  • 10 ml of amylose matrix (NEB) was added to this cleared lysate and mixed by end-over-end rotation overnight at 4 C.
  • the amylose beads were washed 5 times in 50 ml of column buffer before the MBP fusion protein was specifically eluted with an excess of free maltose (10 mM in column buffer, 5 ml total).
  • Protein concentration in the eluate was determined with the BCA kit (Pierce, Rockford, IL), and the purity of the fusion protein was assessed by SDS-polyacrylamide gel electrophoresis ("SDS-PAGE”) and staining with Coomassie Brilliant Blue G250.
  • Immunization with fusion protein MBP-NBC-5 Two guinea pigs (gp) and two rabbits (rab) were subjected to the following immunization protocol: day 0: 1 ml preimmune bleed; day 1 : subcutaneous (s.c.) injection of purified MBP-NBC-5 in complete Freund ' s adjuvant (gp: 20 ⁇ g; rab: 50 ⁇ g); day 28: boost with MBP-NBC5 in incomplete Freund's adjuvant (gp: 20 ⁇ g; rab: 50 ⁇ g); day 38: 2-ml test bleed #1; day 56: boost (as on day 28); day 66: 2- ml test bleed #2. Subsequently, animals were boosted and bled in 4 week-intervals. Antisera were stored at 4 C with 0.03%> sodium azide to prevent microbial growth. Characterization of Antisera by Heterologous Expression.
  • HEK-293 cells For transient expression in HEK-293 cells, the original rNBC cDNA clone, including 5 ' and 3 ' untranslated regions, was cloned into the NotI site of pSV-SPORT-1 (GIBCO). Near-confluent HEK-293 grown on coverslips were transfected using the DEAE-dextran method, and grown for 48 h. Untreated cells, mock-transfected cells, and cells transfected with only the "empty" vector were used as controls. We studied expression of rNBC by indirect immunofluorescence microscopy.
  • cells were fixed in 3%> paraformaldehyde/PBS, permeabilized in 0.3% Triton-X-100/PBS, blocked in 20% goat serum/PBS, and incubated for 1 h at room temperature with the respective sera diluted 1 : 100 in 20%> goat serum/PBS. After washing the sample three times in PBS, we incubated it for 1 h with a rhodamine conjugate of a goat anti-rabbit IgG antibody (Zymed Laboratories Inc., CA), diluted 1 :2,000 in 20% goat serum/PBS.
  • a rhodamine conjugate of a goat anti-rabbit IgG antibody Zymed Laboratories Inc., CA
  • cRNAs coding for rNBC and for murine anion exchanger isoform AE-2 were transcribed from pTLN-2 or pBluescript plasmid vectors, respectively, and injected into stage V-VI oocytes ofN. laevis. Expression of the respective ion transporters was assessed eight days later by monitoring membrane potential and intracellular pH (pHj) changes in response to removal of external Na + and Cl " using microelectrodes. Subsequently, Triton-X-100 extracts of individual oocytes were prepared as described elsewhere, except that methionine was omitted from the extraction buffer. The extracted proteins were separated by SDS-PAGE and immunoblotted as described below.
  • the kidneys were placed in ice-cold homogenization buffer ("HB"; in mM: 250 sucrose, 20 HEPES, pH 7.4 with HCI, 100 NaCl, 2 Na-EDTA, 1 PMSF, 0.001 leupeptin, 0.001 pepstatin) and homogenized using a Polytron).
  • the homogenate was centrifuged (15 min at 3,000 ⁇ m, SS-34 rotor, 4 C) and the pellet (PI), containing debris and nuclei, was discarded.
  • the supernatant (SI) was recentrifuged (45 min at 15,000 ⁇ m, SS-34 rotor, 4 C) and the supernatant (S2) discarded.
  • proteins were transferred overnight at 0.5 - 1.0 mA/cm ⁇ in a semi-dry blotting apparatus (BIO-RAD) onto PVDF membranes (Immobilon-P, Millipore) using the discontinuous Tris/glycine buffer system described by the manufacturer (Millipore).
  • Proteins on the membranes were stained with Coomassie Blue G250, photocopied, and subjected to the immunodetection protocol. All estimates of molecular weight (MW) were obtained by comparison to unstained standards, spaced in regular 10-kDa intervals
  • Solubilization of the native Na + /HCO3" cotransporter from rat kidney To test for the ability of various detergents to solubilize NBC from rat kidney, rat kidney microsomes were treated at room temperature with CHAPS, Tween-80, Tween-20, Triton-X-100, CTAB, Octylglucoside, Nonidet-P-40, 4% sodium dodecyl sulfate (SDS), lithium dodecylsulfate, and 300 mM aminocaproic acid.
  • insoluble material was pelleted by centrifugation at 15,000 ⁇ m for 1 hour at +4°, and aliquots of supernatant and pellet were subjected to the standard immunoblotting protocol, using rabbit anti-(MBP-NBC-5) serum at a dilution of 1 :400.
  • Salamander Female specimens of the aquatic phase Ambystoma tigrinum, kept at 4 C, were anesthetized by submersion in 0.2%> tricaine methanesulfonate. The abdomen was opened via two paramedian incisions and one transverse suprapubic incision.
  • kidneys Following removal of the large intestine, the kidneys were exposed and both kidneys were perfused for 15 minutes via the venous portal circulation with cold, amphibian NaCl Ringer buffered with 10 mM HEPES, pH 7.5. The perfiision solution was then switched to a periodate-lysine-paraformaldehyde fixative (PLP; in mM: 8 NalO 60 L-lysine-HCl, 30 Na2HPO4, 4% paraformaldehyde, in phosphate buffered saline of 200 mOsm). The kidneys were then removed, post- fixed for 4-6 hours in the same fixative, washed in PBS, and stored in 0.5% paraformaldehyde in PBS at 4 C.
  • PBP periodate-lysine-paraformaldehyde fixative
  • Rat and rabbit Adult New Zealand White rabbits and Sprague-Dawley rats were anesthetized with sodium pentobarbital, and the kidneys perfusion-fixed by first inserting a cannula into the descending aorta distal to the renal arteries. The kidneys were then perfused retrograde with PBS, pH 7.4 at 37 C, to remove blood, followed by PLP fixative (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate in PBS, pH 7.4). For cryostat sections, kidneys were cut in half on a mid-sagittal plane and post- fixed in PLP for 4-6 hours. The fixed tissue was then cryoprotected overnight in a 30% solution of sucrose in PBS.
  • PLP fixative 2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate in PBS, pH 7.4
  • Indirect Immunofluorescence Microscopy was performed on either 5 ⁇ m cryosections or on 0.5 ⁇ m cryosections. Briefly, tissue sections were washed sequentially in PBS, then in 50 mM NH4CI in PBS, and in blocking buffer (1%> bovine serum albumin in PBS). This was followed by a 1 hour-incubation with the primary antiserum, diluted 1 :50 in 50% goat serum in PBS.
  • Immunoblots of oocytes expressing rkNBC were performed immunoblot experiments on Xenopus oocytes expressing rkNBC. As controls, oocytes were injected with either water or cRNA encoding AE2. AE2 was included as a control because, among all proteins whose primary structure is known, the anion exchangers have the highest degree of amino acid sequence similarity compared to the NBCs. In several oocytes, we used electrophysiological techniques to confirm expression of AE2 or rkNBC. On several others, we performed the immunoblots using either rabbit anti-(MBP-NBC-5) serum or rabbit anti- (MBP-NBC-3) serum.
  • NBC protein in amphibian and mammalian species we performed immunoblotting experiments with rabbit anti-(MBP-NBC-5) serum on renal microsomes from whole kidneys of salamander, rat, and rabbit.
  • the antiserum detected a single band of -160 kDa. This is substantially greater than the 116 kDa predicted from the cDNA sequence of aNBC.
  • the predominant bands were at a MW of -130 kDa.
  • the antiserum also routinely detected minor bands at -100 kDa and -85 kDa; the band below -80 kDa was variable.
  • the antisera In the rabbit kidney, the antisera also routinely detected the -100 kDa band; the lower MW bands were variable.
  • Antibody-preabsorption experiments on rat kidney The specificity of the labeling observed in the kidneys of salamander, rat, and rabbit was tested in further immunoblotting experiments.
  • probed with native rabbit anti-(MBP-NBC-5) serum we observed a major band at -130 kDa, and two minor bands at -100 and -85 kDa.
  • MBP-NBC-5 In a lane labeled MBP-NBC-5, we probed with anti-(MBP-NBC-5) serum that had been depleted of antibodies directed against MBP- NBC-5 (by preincubating with an excess of MBP-NBC-5). We observed no bands whatsoever. These results demonstrate that all three bands observed in lanes 1 - 3 were due to labeling by antibodies specifically directed against the NBC-5 portion of rkNBC. There is no evidence for cross-reactivity of antibodies directed against either MBP per se, or against bacterial contaminants.
  • a -130-kDa band was also observed in Xenopus oocytes heterologously expressing rkNBC, it is likely that the -130 kDa protein truly corresponds to the rat-kidney Na+ HCO 3 cotransporter protein.
  • the nature of the two minor bands at -100 kDa and -85 kDa is not clear. However, the antibody-depletion experiments demonstrate that proteins in these two bands have NBC- specific epitopes.
  • the two bands may represent proteolytic fragments of the -130 kDa protein, or different NBC-isoforms.
  • HCO3 secretion amounts to approx. 10%> of gastric acid secretion and is on of the major protective mechanisms against luminal acid and tryptic enzymes.
  • the cellular mechanisms for HCO3 secretion in the stomach are poorly understood, and little data are available on mammalian species.
  • Our immunoblotting experiments show that rat stomach expresses a Na + HCO3" cotransporter of similar molecular weight as the renal form. The specificity of the labeling of this protein is confirmed by antibody preabso ⁇ tion experiments. The antibody titering experiment demonstrated the relatively high affinity of the antiserum for this protein.
  • Immunolocalization of NBC in rat, rabbit, and salamander kidney To determine the cellular and subcellular location of the NBC protein in the kidneys of rat, rabbit and salamander, we performed immunofluorescence staining of semi-thin (0.5 mm) sections, and immunoperoxidase staining of standard (5 mm) PLP-fixed cryosections. We used guinea-pig anti-(MBP-NBC-5) serum in both cases. For each tissue, we separately assessed the specificity of the antiserum, using procedures analogous to those described above for the immunoblots. Using preimmune serum, we observed no staining, a finding that rules out the presence of antibodies against cytoskeleton components that are sometimes spontaneously found in rabbit serum.
  • the rabbit kidney exhibited the same staining pattern as we observed for the rat kidney, using the guinea-pig anti-(MBP-NBC-5).
  • Polyclonal antisera was obtained from guinea pigs immunogenized with a fusion antigen of MBP to a fragment of NBC, residues 338-391 (GP3) or residues 928-1035 (GP5), and was used to probe western blots of crude membranes isolated from Ambystoma tigrinum, rat and rabbit ( Figure 21). Summary. We have demonstrated the ability of the utilized antisera to detect NBC proteins from amphibian (e.g., salamander) and mammalian (e.g., rat, rabbit, guinea pig and human) species.
  • amphibian e.g., salamander
  • mammalian e.g., rat, rabbit, guinea pig and human
  • Northern blot A human multiple tissue Northern blot (catalog number 7760-1) was purchased from Clontech (Palo Alto, CA). 32 P-labeled, random-hexamer-primed cDNA probe was made from the unique 5' region of the hhNBC (+7 to +271). Hybridization was performed in the ExpressHyb (Clontech) hybridization buffer at 68°C for two hours, with the probe concentration of 0.83 x 10 6 cpm/ml. The membranes were washed at 37°C in 2 ⁇ SSC/0.1% SDS for 40 min and then at 50°C in 0.1 x SSC/0.1% SDS for 1.5 hours, and then autoradiographed for 7 days.
  • Northern blot The 3' region (2227-3063) of raNBC were random-primed using the Random priming kit (GibcoBRL).
  • the multiple tissue Northern blots for rat or human were purchased from Clontech (Palo Alto, CA). Hybridization was performed in the ExpressHyb hybridization buffer (Clontech) at 68°C for one hour, with 1-2 x 10 6 cpm/ml. The membrane was then washed at room temperature in IX SSC/0.1% SDS for 40 min, and at 50°C in 0.1X SSC/0.1%) SDS for 1.5 hours. The blot was then autoradiographed for 24 hours. Results
  • the radiolabeled raNBC probe detects strong signals in rat spleen (-7.5 kb) and at moderate levels in heart and kidney. At low level, brain, lung, liver also express raNBC. In testis, raNBC detects a smaller (-4.1 kb) transcript. The -7.5 kb transcript is also weakly present in testis.
  • the radiolabeled NT2 probe detects strong signals in human peripheral blood leucocytes, testis, spleen, and skeletal muscle (-9 kb). The signal is also detectable in other tissues such as colon, placenta, kidney, and heart. In contrast to raNBC, which detects a major 4.1 kb transcript in testis, NT2 detects the same 9 kb transcript. The -5 kb transcript is also weakly present in testis.
  • Oocyte experiments Oocytes of Xenopus laevis were obtained as described in Romero et al. (1997) using collagenase (type IA; Sigma). The open reading frame of hhNBC was ligated to the EcoRI site of pGH19. Plasmids encoding hhNBC cDNAs were linearized with NotI prior to transcription, and then in vitro transcribed with the mMessage mMachine kit (Ambion, Austin, TX) using T7 RNA polymerase. To obtain a maximum production of a full length transcript, the ratio of cap analog to GTP was decreased by increasing the amount of GTP to 3 mM final.
  • the defolliculated oocytes (Stage V and VI) were injected with 50 nl of RNA (0.5 mg/ml) or water, and incubated in the OR3 buffer (50% Leibovitz L-15 media with L-glutamine, 5 mM HEPES, pH 7.5) supplemented with 5 U/ml penicillin/Streptomycin. Injected oocytes were maintained for 3-7 days at 18°C before use.
  • a borosilicate glass capillary (1.5 mm OD, Frederick Haer Co., MD) was pulled, dried in an oven at 200°C for at least 2 h, and vapor silanized with bis(dimethylamino)-dimethyl silane (Fluka) in a closed vessel.
  • the electrode tip was then filled with hydrogen ionophore 1 cocktail B (Fluka), and back filled with phosphate buffer (pH 7.0) (Amman, 1981) connected to a high-impedance electrometer (FD-223, WPI, Ine) and calibrated in standard solutions of pH 6 and 8.
  • the slope was generally 55-57 mV/pH unit.
  • the standard ND96 contained 96 mM NaCl, 2 mM KCl, 1 mM MgC12, 1.8 mM CaCl 2 , and 5 mM HEPES (pH 7.5).
  • the 1.5% C0 2 / 10 mM HCO>equilibrated solution was by substituting 10 mM HCO ⁇ 3 for NaCl to maintain a constant ionic strength.
  • the Na + substitute was choline.
  • the Cl " substitute was gluconate.
  • Oocytes expressing hhNBC were assessed to HCO ⁇ 3 transport by monitoring the recovery of intracellular pH (pHj) during 1.5% CO 2 /10 mM HCO ⁇ 3 exposure.
  • Applying CO 2 /HCO " 3 causes a slow and sustained decrease in pHj in oocytes injected with hhNBC-cRNA.
  • the CO 2 /HCO ⁇ 3 caused rapid hype ⁇ olarization in oocytes expressing hhNBC (n>6), whereas modest, slowly developing depolarization in water-injected oocytes.
  • Plasmids encoding NT2 cDNA were linearized with NotI prior to transcription, and then in vitro transcribed with the mMessage mMachine kit (Ambion, Austin, TX) using T7 RNA polymerase. The final concentration of GTP was 3 mM, which would increase the transcription efficiency to obtain full length transcript.
  • the defolliculated oocytes (Stage V and VI) were injected with 50 nl of RNA (0.25 mg/ml) or water, and incubated in the OR3 buffer (50%) Leibovitz L-15 media with L-glutamine, 5 mM HEPES, pH 7.5) supplemented with 5 U/ml penicillin/Streptomycin.
  • Injected oocytes were maintained for 4-6 days at 18°C before use.
  • the pH electrode was prepared as described previously. Solutions.
  • the standard ND96 contained 96 mM NaCl, 2 mM KCl, 1 mM MgC12, 1.8 mM
  • electrogenic N--/ ⁇ CO3 cotransporter has 3 separate binding sites for
  • NBC-like clones that are identified herein or will be identified using the procedures set forth herein may transport other ions.
  • hPanK-NBC was generated by Reverse transcriptase-PCR (RT-PCR), followed by 30 cycles of PCR using human NBC-specific primers.
  • Human pancreas poly(A)+ RNA was purchased from Clontech (Palo Alto, CA). 500 nanogram of human pancreatic poly(A)+ RNA was reverse transcribed using Superscript II- RT (Gibco-BRL, Gaithersburg, MD) according to manufactures instructions.
  • Method steps include: (a) 500 ng + oligo-dT to 12 uL, heat to 70 C for 10 min; (b) ice for > 1 min; (c) buffer, Mg++, dNTPs, and DTT to 19 uL; (d) mix; (e) incubate at 42 C for 5 min; (f) add 1.OuL Superscript II-RT (200U); (g) mix with pipette; (h) incubate at 42 C for 55 min; (i) incubate at 70 C for 15 min to inactivate; and, (j) incubate at 4 C; and, (k) store at -20 C. PCR- Amplification.
  • Method steps include: (a) 0.5 ul of RT in 50 uL rxn; (b) 25 uM of SSSS_f and ERTHSC_r; (c) 1 cycle(94 C for 2 min, 60 C for 2 min, 68 C for 2 min); (d) 30 cycles of (94 C for 20 sec, 60 C for 30 sec, 68 C for 3.5 min); (e) 1 cycle(68 C for 15 min, 4 C for 24 hr); and, (f) store at -20 C.
  • nucleotide sequence and amino acid sequence of hpNBC is provided in Figure 27.

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Abstract

L'invention porte sur des séquences nucléotidiques de molécules d'acide nucléique codant pour des protéines membres de la famille des 'cotranporteurs du bicarbonate de soude' (NBC). Dans le cadre de cette découverte, l'invention porte également sur des protéines de NBC isolées, sur des molécules d'acide nucléique isolées codant pour le NBC, sur des procédés d'isolement des molécules d'acide nucléique codant pour d'autres protéines membres de la famille des NBC, sur des procédés d'identification d'agents agonistes ou antagonistes de l'activité des NBC modulant certains processus biologiques ou pathologiques, et sur des procédés de contrôle de l'activité des NBC.
PCT/US1998/010297 1997-05-20 1998-05-20 Le nbc, gene codant pour un membre la famille de proteines transporteuses de bicarbonate WO1998053067A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037637A1 (fr) * 1998-12-21 2000-06-29 Takeda Chemical Industries, Ltd. Proteine et adn associe
EP0897982A3 (fr) * 1997-08-19 2000-12-06 Smithkline Beecham Laboratoires Pharmaceutiques Co-transporteurs de bicarbonate de sodium
WO2001059446A3 (fr) * 2000-02-11 2002-03-14 Biofocus Discovery Ltd Analyse amelioree
WO2002040542A3 (fr) * 2000-11-20 2004-02-26 Millennium Pharm Inc 58569 et 50111, proteines humaines et leur methodes d'utilisation
US7183075B2 (en) 2000-04-14 2007-02-27 Vanderbilt University Purified and isolated potassium-chloride cotransporter nucleic acids and polypeptides and therapeutic and screening methods using same

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BORON V F ET AL: "The renal electrogenic Na +:HCO-3 cotransporter.", JOURNAL OF EXPERIMENTAL BIOLOGY, (1997 JAN) 200 ( PT 2) 263-8. REF: 24 JOURNAL CODE: I2F. ISSN: 0022-0949., ENGLAND: United Kingdom, XP002076892 *
CHARLES E. BURNHAM ET AL.: "Cloning and functional expression of a human kidney Na+:HCO3- Cotransporter", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 31, 1 August 1997 (1997-08-01), MD US, pages 19111 - 19114, XP002076894 *
MICHAEL F. ROMERO ET AL.: "Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter", NATURE, vol. 387, 22 May 1997 (1997-05-22), LONDON GB, pages 409 - 413, XP002076896 *
ROMERO M F ET AL: "Cloning and functional expression of rNBC, an electrogenic Na (+)- HCO3 - cotransporter from rat kidney.", AMERICAN JOURNAL OF PHYSIOLOGY, (1998 FEB) 274 (2 PT 2) F425-32. JOURNAL CODE: 3U8. ISSN: 0002-9513., United States, XP002076895 *
ROMERO M F ET AL: "Cloning and functional expression of the rat renal electrogenic Na - HCO3 cotransporter (rNBC).", 29TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF NEPHROLOGY, NEW ORLEANS, LOUISIANA, USA, NOVEMBER 3-6, 1996. JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 7 (9). 1996. 1259. ISSN: 1046-6673, XP002076893 *
Trrod Database Entry O35422 Accession number O35422; 1 January 1998 BURNHAM C.E. ET AL. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0897982A3 (fr) * 1997-08-19 2000-12-06 Smithkline Beecham Laboratoires Pharmaceutiques Co-transporteurs de bicarbonate de sodium
WO2000037637A1 (fr) * 1998-12-21 2000-06-29 Takeda Chemical Industries, Ltd. Proteine et adn associe
US6723522B1 (en) 1998-12-21 2004-04-20 Takeda Chemical Industries, Ltd. Sodium bicarbonate cotransporter protein
WO2001059446A3 (fr) * 2000-02-11 2002-03-14 Biofocus Discovery Ltd Analyse amelioree
US7183075B2 (en) 2000-04-14 2007-02-27 Vanderbilt University Purified and isolated potassium-chloride cotransporter nucleic acids and polypeptides and therapeutic and screening methods using same
WO2002040542A3 (fr) * 2000-11-20 2004-02-26 Millennium Pharm Inc 58569 et 50111, proteines humaines et leur methodes d'utilisation

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