WO2001083809A2 - Cell membrane nucleic acid transport channel complex - Google Patents

Abstract

A cell membrane nucleic acid channel protein complex is a heteromultimer of a 45 kDa cell membrane channel forming protein and a 36kDa regulatory protein, wherein the regulatory protein regulates the specificity of the channel forming protein for nucleic acid transport.

Description

CELL MEMBRANE NUCLEIC ACID TRANSPORT CHANNEL COMPLEX

This application is based on U.S. Provisional Application Serial No. 60/200,680.

The research leading to the present invention was supported, in part, by National Institute of Diabetes, Digestion and Kidney/National Institutes of Health Grant No. 5P01DK50795. Accordingly, the U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a novel nucleic acid channel protein complex in cell membranes which selectively conducts nucleic acids into a cell.

BACKGROUND OF THE INVENTION

Nucleic Acid Intemalization by Cells

Nucleic acid uptake by most tissues and cells in vivo is relatively low. Nucleic acid intemalization is highest in the kidney, liver, and brain, and relatively low or undetectable in other tissues studied (Oberbauer R et al., Kidney Int, 1995, 48: 1226-1232; Rappaport J et al., Kidney Int, 1995, 47:1462-1469; Ogawa S et al., Regul Pept, 1995,

59:143-9; Meeker R, et al., J Neuroendocrinology, 1995, 7:419-428; and Cossum PA et al., J Pharmacol Exp Ther, 1993, 267: 1181-1190). However, the brain is not a site of significant accumulation following systemic administration of nucleic acids since nucleic acids do not readily cross the blood brain barrier. In the liver, oligodeoxyribonucleic acids (ODN) are internalized by hepatocytes; breakdown of the nucleotides begins within minutes after entering the cell. ODN are completely degraded in the liver within hours of a single bolus injection (Agrawal S et al., Proc Natl Acad Sci, 1991, 88:7595-7599; Goodarzi G et al., Biopharm Drug Dispos, 1992, 13:221-227). In the kidney, breakdown of ODN is not seen for up to an hour, and full length ODN can be detected in kidney tissue and urine for days after a single bolus injection of labeled ODN (Agrawal et al., 1991; Rappaport J et al.,1995; Oberbauer R et al., 1995). The unique handling of ODN by kidney (i.e., uptake by cells without rapid degradation) makes the kidney a prime site of nucleic acid toxicity. The molecular mechanism of oligonucleotide uptake is not well understood.

Several mechanisms of ODN uptake have been proposed, including receptor mediated endocytosis (Stein CA et al., Biochemistry, 1993, 32:4855-4861; Krieg AM et al., Proc Natl

Acad Sci USA, 1993, 90:1048-1052; Wu-Pong et al., Pharm Res, 1992, 9:1010-1017), and fluid phase endocytosis, i.e., pinocytosis (Yakubov LA et al., Proc Natl Acad Sci, 1989,

86:6454-6458; Stein CA et al., Biochemistry, 1993, 32:4855-4861; Vlassov et al., Biochim

Biophys Acta, 1994, 1197: 95-108; Gao et al., 1993).

Nucleic acid uptake by endocytosis or pinocytosis, however, may not be specific for nucleic acids. Pinocytosis does not have a mechanism that is specific for nucleic acid binding. ODN uptake in these models can be explained as a "bystander" effect, that is,

ODN adsorb to the cell surface via a relatively non-specific interaction with a cell surface protein, and are internalized during normal membrane turnover.

Another mechanism for ODN uptake is through a cell membrane nucleic acid conducting channel. A protein that forms this channel, isolated from rat kidney brush border membrane, selectively binds single-stranded nucleic acid sequences (Hanss B et al., Proc Natl

Acad Sci, 1998, 95:1921-1926). When reconstituted in lipid bilayers this 45 kDa protein functioned as a gated channel, allowing the passage of nucleic acids through the channel.

However, the in vivo mechanism for regulating the function of this protein has heretofore not been discovered. It is desirable to find a method to modify or manipulate the transport of nucleic acids into cells, particularly renal epithelial cells, in order to treat or provide therapy for conditions in which nucleic acids are involved, for example, to protect the kidney by preventing deleterious accumulation of oligonucleotides as a result of an oligonucleotide- based therapy.

SUMMARY OF THE INVENTION

The present invention provides a novel cell membrane nucleic acid channel protein complex comprising a first subunit protein and a second subunit protein. The first subunit protein of the nucleic acid channel protein complex of the invention is a purified cell membrane channel forming protein, p45, having a molecular weight of approximately 45kDa and comprising the amino acid sequences NVHWAGSDSK (SEQ ID NO:l), XTATEXSTYATNK (SEQ ID NO:2), KQEEAQLKQIADA (SEQ ID NO:3) and DASCRLFDRAD (SEQ ID NO:4), wherein X is I or L.

The second subunit protein of the nucleic acid channel protein complex is a regulatory protein that regulates the specificity of the channel forming protein for nucleic acid transport. In one embodiment the regulatory protein is cytosolic malate dehydrogenase. Preferably, the regulatory protein has the amino acid sequence of SEQ ID NO: 5.

In one aspect of the invention, the cell membrane is located in a kidney cell. In another embodiment, the cell membrane is not a kidney cell membrane, but may be a membrane in a cell from another organ. In another embodiment, the cell membrane is part of a whole intact cell.

The present invention further provides a method of regulating the transport of nucleic acids into a cell. In one aspect of the invention, the method comprises modifying the activity of a purified cell membrane channel forming protein comprising the amino acid sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 4 to have nucleic acid specific transport activity. In one aspect of this method, a cell is modified to express a protein that regulates the specificity of the cell surface channel forming protein so that it selectively transports nucleic acids across the membrane into the cell. In one embodiment, the regulatory protein is cytosolic malate dehydrogenase. In a preferred embodiment, the regulatory protein has the amino acid sequence of SEQ ID NO: 5.

The present invention further provides a method of producing a cell having a nucleic acid transport channel protein complex. The method comprises introducing into the membrane of a cell a purified cell membrane channel forming protein comprising the amino acid sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 4. The method further comprises introducing into a cell membrane a protein that regulates the specificity of the cell surface channel forming protein so that it selectively transports nucleic acids through the cell membrane into the cell (i.e., a regulatory protein). In one embodiment, the regulatory protein is cytosolic malate dehydrogenase. In a preferred embodiment, the regulatory protein has the amino acid sequence of SEQ ID NO: 5. In another aspect, the present invention provides a method of identifying compounds that modulate the activity of a cell membrane nucleic acid transport channel protein complex. In this aspect, the method comprises detecting the activity of the nucleic acid transport channel protein complex in the presence of a test compound. The test compound may be an agonist or antagonist of channel protein complex activity. The test compound may be an inhibitor of one of the subunits of the protein complex. In one embodiment, the compound blocks the activity of cytosolic malate dehydrogenase.

In a further aspect, the present invention provides a method of reducing nucleic acid toxicity in kidney cells. In this aspect, the method comprises administering an agent that inhibits or reduces the activity of a nucleic acid transport channel protein complex. The activity may be reduced or inhibited such that the transport of nucleic acids into the cell is reduced or inhibited. In one embodiment, the agent blocks cytosolic malate dehydrogenase activity.

DESCRIPTION OF THE FIGURES

Figures 1A, 1B,1C and ID show silver stained SDS-PAGE analysis of fractions from the purification of the nucleic acid channel proteins that have nucleic acid channel activity. Figure 1A - Total renal brush border membrane protein, isolated by differential and density gradient centrifugation. Figure IB - Protein content of combined fractions containing nucleic acid dependent channel activity after two steps of ion-exchange chromatography. Figure 1C - Hydrophobicity chromatography resulted in a single active peak which contained two protein bands at 36 kDa and 45 kDa. Figure ID - Pure p45. Figures 2A, 2B, 2C, 2D, 2E, and 2F show current output traces demonstrating functional reconstitution of nucleic acid channel subunits. Proteoliposomes were formed with either pure p45 or pure p36 and were fused either individually or jointly with planar lipid bilayers. The solid horizontal line indicates zero current. Holding potentials are shown for each trace. Figure 2A - Current trace of reconstituted affinity purified nucleic acid channel as described in Hanss B et al., 1998 and Leal-Pinto et al., 1996. Figure 2B - Reconstitution of pure p45 in the absence of oligodeoxyribonucleic acids (ODN). Purified p45 conducted current in the absence of ODN indicating pure p45 alone forms a non-selective transmembrane channel. Figure 2C - Channel activity observed following addition of 10 μM ODN to reconstituted pure p45. Addition of ODN resulted in fluctuations of current but did not alter channel gating. Figure 2D - Reconstitution of pure p36 in the absence of ODN. No current was observed. Figure 2E - Reconstitution of pure p36 with 10 μM ODN. Current was not observed when pure p36 was reconstituted either in the absence (D) or presence (E) of ODN, indicating that p36 did not form a channel. Figure 2F - Reconstitution of pure p45 and pure p36 in the presence of 10 μM ODN. Reconstitution of both pure p45 and p36 restored the nucleic acid dependence of the channel and altered gating kinetics.

Figure 3 shows a comparison of the sequence of p36 tryptic peptides VIVVGNPANTNCLTASK (SEQ ID NO:6), ENFACLTR (SEQ ID NO: 7), LGVTADDVK (SEQ ID NO: 8), NVIIWGNHSSTQYPDVN (SEQ ID NO:9), GEFITTVQQR (SEQ ID NO: 10) and FVEGLPINDFSREK (SEQ ID NO: 11) and human cytosolic malate dehydrogenase (cMDH) (SEQ ID NO:5).

Figures 4A and 4B show structural analysis of human cMDH. Figure 4A shows hydropathy analysis (Kyte-Doolittle) of human cMDH. Figure 4B shows the predicted transmembrane helixes in human cMDH.

Figures 5A, 5B and 5C show the current output traces from an experiment investigating the ability of L-malate, the primary substrate for cMDH, to block nucleic acid channel activity. The solid horizontal line indicates zero current. Membrane potential was held at +50 mV for all traces. Figure 5A - Control activity (no L-malate present). Open probability under these conditions was 0.36. Figure 5B - Current output trace following addition of 5 μM L-malate. This malate concentration resulted in a 72% reduction in channel open probability. Figure 5C - Current trace in the presence of 75 μM L-malate; this concentration of L-malate resulted in complete blockade of nucleic acid channel activity.

Figures 6 A, 6B, 6C, 6D and 6E show the results of the analysis of the antiserum raised against cMDH. Figure 6A - ELIS A of serum from a mouse immunized with cMDH (Sigma Chemical Co). The filled bar represents normal mouse serum. Figure 6B - Westem blot of pig-heart cMDH. Figure 6C - Westem blot of whole kidney lysate. Figure 6D and Figure 6E - Current output traces from an experiment in which antiserum against cMDH was used to block nucleic acid channel activity.

Figures 7A, 7B, 7C and 7D show current output traces demonstrating functional reconstitution of nucleic acid channel activity with purified p45 and pig-heart cytosolic malate dehydrogenase. Zero current is indicated by the solid horizontal line.

Membrane potential was held at +50 mV for each trace. Figure 7A - Reconstitution of pure p45 in the absence of ODN demonstrated ODN independent channel activity. Figure 7B - Reconstitution of pig-heart cMDH; ODN were not present; channel activity was not seen in the absence or presence of ODN (shown in B). Figure 7C - Reconstitution of pure p45 and pig-heart cMDH in the presence of ODN; channel activity was not seen under these conditions. Figure 7D - Reconstitution of pure p45 and pig-heart cMDH in the presence of 10 μM ODN; reconstitution of pure p45 and commercially available pig-heart cMDH recovered the activity of the nucleic acid channel purified from rat-kidney brush border membrane.

DETAILED DESCRIPTION OF THE INVENTION The channel protein complex of the present invention forms a gated channel in a cell membrane that selectively transports nucleic acids across the membrane. Applicants have found that by modulating the activity of the protein complex, transportation of nucleic acids across the membrane can be manipulated. In one aspect of the invention, the protein complex may be modified such that nucleic acids are no longer transported across the membrane, or the transport of nucleic acids across the membrane is reduced or inhibited.

Accordingly, in a whole intact cell, altering the properties of this nucleic acid channel protein complex provides a vehicle for modulating the passage of nucleic acids into cells for studying the effect of these molecules on the cells. In such a manner, conditions in which the transport and accumulation of nucleic acids into a cell would be deleterious to the cell or host system may be ameliorated.

The present invention is based in part on the purification and characterization of the subunits of a nucleic acid transport channel complex. A 45 kDa nucleic acid binding protein was initially isolated from renal proximal brush border membrane (Hanss B et al., 1998). The 45 kDa protein was reconstituted in a lipid bilayer model and tested for nucleic acid transport function. Nucleic acid transport activity was seen when ODN were added to the system and the channel activity was correlated to the presence of the 45 kDa protein. Applicants unexpectedly found, upon purification under reducing conditions described below, that the fractions containing the 45 kDa protein and having nucleic acid channel transport activity contained a 45 kDa protein, the p45, and a second protein with an approximate molecular weight of 36kDa, the p36. When reconstituted alone, the p36 had no channel activity in the lipid bilayer model. However, when the p36 was reconstituted with pure p45, the p36 converted the pure p45 from an ODN independent channel to a nucleic acid-gated nucleic acid-conducting channel. The regulatory subunit was identified as cMDH. Thus it was discovered that the isolated channel protein comprised a heterodimer complex comprising the 45kDa protein subunit and the 36 kDa protein subunit, the purified p45 and p36, respectively.

In addition, two other proteins were detected in the fractions containing the channel complex. These proteins were identified as regucalcin, known to have a role in signal transduction, particularly of cellular calcium distribution, and calreticulin, a calcium binding protein of the endoplasmic reticulum, which acts as a chaperone for newly synthesized proteins.

According to the present invention, a cell membrane protein that forms a non- selective ion-conducting channel through the cell membrane may be modified such that the membrane protein can selectively transport nucleic acids. In this manner one can regulate the transport of nucleic acids into a cell by modifying a membrane protein to have nucleic acid specific transport activity. For example, a cell that has a membrane protein which in its natural state forms a channel and demonstrates channel activity, i.e., is open in the absence of ODN, such as the pure p45 alone, will become gated selectively open for nucleic acid transport if the cell is modified to express a regulatory protein which renders the pure p45 specific for nucleic acid transport. An example of such a regulatory protein includes cMDH. In one aspect, the cell is modified to express the regulatory protein.

In a further aspect, host cells that do not have nucleic acid transport channel proteins may be modified such that a nucleic acid channel protein complex is formed in the cell, making the cells accessible to nucleic acids. The identification of these proteins and their ability to form a channel complex for transporting nucleic acids provides the means for mediating passage of nucleic acids across cell membranes into cells.

The identification of these proteins also allows for the identification of compounds that can modulate the activity of the channel complex by modifying the activity of one or both of the subunit proteins of the channel complex.

Definitions The term "nucleic acid transport channel protein complex" is used herein to mean a protein complex having a first protein subunit which is a transport channel forming protein and a second protein subunit which is a transport channel regulatory protein. The protein complex is typically located in a cell membrane or in a cell membrane model, i.e., planar lipid bilayer model.

The term "transport channel forming protein" is used herein to mean a purified protein of approximately 45 kDa molecular weight and having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. The purified 45 kDa protein subunit, substantially free of the second protein subunit is also referred to herein as "p45" or "pure p45".

The term "regulatory protein" is used herein to mean a protein that regulates the specificity of the activity of a channel forming protein to transport nucleic acids through the channel. The regulatory protein is of approximately 36 kDa molecular weight and is substantially free of the first subunit protein and is also referred to herein as "p36" or "pure p36" Examples of such regulatory proteins include cytosolic malate dehydrogenase (cMDH). In particular the regulatory protein the amino acid sequence of SEQ ID NO: 5.

A nucleic acid or polypeptide sequence that is "derived from" a designated sequence refers to a sequence that corresponds to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are homologous or complementary to the sequence, as well as "sequence-conservative variants" and "function-conservative variants."

"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. "Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Clustal Method, wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide or enzyme which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared. Finally, for purposes of the invention, a functional-conservative variant includes a truncated form of the protein that performs its function, or proteolytic fragments of the nucleic acid transport channel protein complex. Functional-conservative variants also include any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide.

Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80% of the amino acids are identical, or greater than about 90% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of the programs described above (BLAST, FASTA, MACAW).

As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of about 10 or more nucleotides, preferably of about 20 to 50 or more nucleotides, and more preferably of about 100 to 150 nucelotides. Oligonucleotides can be labeled, e.g., with ³²P- nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc. The present invention provides for transport of nucleic acids or oligonucleotides into cells which may be used to inhibit expression of a target protein. Specific non-limiting examples of synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH₂-NH-O-CH₂, CH₂-N(CH₃)-O-CH₂, CH₂-O-N(CH₃)-CH₂, CH₂-N(CH₃)-N(CH₃)-CH₂ and O-N(CH₃)-CH₂-CH₂ backbones (where phosphodiester is O- PO₂-O-CH₂). US Patent No. 5,677,437 describes heteroaromatic olignucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Patents No. 5,792,844 and No. 5,783,682). US Patent No. 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds. Also envisioned are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al., Science, 1991, 254:1497). Other synthetic oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH₃, F, OCN, O(CH₂)_nNH₂ or O(CH₂)_nCH₃ where n is from 1 to about 10; C, to C*₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-; S-, or N-alkyl; O-, S-, or N- alkenyl; SOCH₃ ; SO₂CH₃; ONO₂;NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group. Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine, such as inosine, may be used in an oligonucleotide molecule.

As used herein, the term "isolated" means that the nucleic acid transport channel protein complex is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non- regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.

Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid.

An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane- associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

Protein purification methods are well known in the art and a specific example of a method for purifying the subunits of the nucleic acid transport channel protein complex is provided in the examples below. For example, nucleic acids can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means. Polypeptides and proteins can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting- out chromatography, extraction, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting (FACS)). Other purification methods are possible. A purified material may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. The term "substantially pure" indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.

Proteins purified by the various techniques described herein can be characterized by methods known in the art including SDS-PAGE, size exclusion chromatography, and electron microscopy and immunodetection.

In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. Alternatively, particularly in biology, the term "about" can mean within an order of magnitude of a given value, and preferably within one-half an order of magnitude of the value.

Molecular Biology Definitions In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); ,4w^'mα/ Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B.EPerbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

A "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxy cytidine; "DNA molecules"). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described infra.

A "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. An "opening reading frame" (ORF) as used herein is a region of a polynucleotide sequence having a start and codon and which may encode a polypeptide. This region may represent a portion of a coding sequence or may comprise a total coding sequence for the polypeptide. A "complement" of a nucleic acid sequence as used herein refers to the

"antisense" sequence that participates in Watson-Crick base-pairing with the original sequence.

The term "gene", also called a "structural gene" means a DNA sequence that codes for or corresponds to a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes. A gene as used herein may or may not include non- transcribed regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Furthermore, a transcribed portion of the gene may include 5'- and 3 '-untranslated sequences and introns in addition to the coding sequence. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A coding sequence is "under the control" or "operatively (or operably) associated with" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence. The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself , e.g. the resulting protein, may also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular or secreted. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside a cell. A substance is "secreted" by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.

The term "transfection" means the introduction of a foreign nucleic acid into a cell. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone." The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.; they are discussed in greater detail below.

The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Expression systems include mammalian host cells and vectors. Suitable cells include C12 cells, CHO cells, HeLa cells, 293 and 293T (human kidney cells), mouse primary myoblasts, and NIH 3T3 cells.

Vectors A wide variety of host/expression vector combinations may be employed in expressing DNA sequences either subunit of the nucleic acid transport channel protein complex, or inhibitors of one or both subunits of the nucleic acid transport channel protein complex, such as antisense nucleic acids or anti-nucleic acid transport channel protein complex intracellular antibodies. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene, 1988, 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Expression of the protein or polypeptide may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature, 1981, 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell, 1980, 22:787-797), the herpes thymidine kinase promoter (Wagner, et al., Proc Natl Acad Sci USA, 1981, 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature, 1982, 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff, et al., Proc Natl Acad Sci USA, 1978, 75:3727-3731), or the tac promoter (DeBoer, et al., Proc Natl Acad Sci USA, 1983, 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American,

1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter and alkaline phosphatase promoter.

A vector can be introduced in vivo in a non- viral vector, e.g., by lipofection, with other transfection facilitating agents (peptides, polymers, etc.), or as naked DNA. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection, with targeting in some instances (Feigner, et al., Proc Natl Acad Sci USA, 1987, 84:7413-7417; Feigner and Ringold, Science, 1989, 337:387-388; see Mackey, et al., Proc Natl Acad Sci USA, 1988, 85:8027-8031; Ulmer et al., Science, 1993, 259:1745-1748). Useful lipid compounds and compositions for transfer of nucleic acids are described in International

Patent Publications WO95/18863 and WO96/17823, and in U.S. Patent No. 5,459,127. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g. , International Patent Publication WO96/25508), or a cationic polymer (e.g. , International Patent Publication WO95/21931 ). A relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir et al., CP Acad Sci, 1998, 321 :893; WO 99/01157; WO 99/01158; WO 99/01175). DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, micro injection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection), or use of a DNA vector transporter (see, e.g., Wu et al, J Biol Chem, 1992, 267:963-967; Wu and Wu, J Biol Chem, 1988, 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990; Williams et al., Proc Natl Acad Sci USA, 1991, 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum Gene Ther, 1992, 3:147-154; Wu and Wu, J Biol Chem, 1987, 262:4429-4432). US Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal.

Also useful are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional protein or polypeptide (as set forth above) can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995. Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 1992, 7:980-990). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl) vector (Kaplitt et al., Molec Cell Neurosci, 1991, 2:320- 330), defective herpes virus vector lacking a glycoprotein L gene (Patent Publication RD 371005 A), or other defective herpes virus vectors (International Patent Publication No. WO 94/21807, published September 29, 1994; International Patent Publication No. WO 92/05263, published April 2, 1994); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J Clin Invest, 1992, 90:626-630; see also La Salle et al., Science, 1993, 259:988-990); and a defective adeno-associated virus vector (Samulski et al., J Virol, 1987, 61:3096-3101; Samulski et al., J Virol, 1989, 63:3822-3828; Lebkowski et al., Mol Cell Biol, 1988, 8:3988-3996).

Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

Cytosolic Malate Dehydrogenase

Malate dehydrogenase is a highly conserved, ubiquitous enzyme present in bacteria, plants, and animals. In mammalian cells there are two isoforms of malate dehydrogenase; a mitochondrial isoform (mMDH) and a cytosolic isoform (cMDH). Both isoforms are NAD-dependent enzymes that catalyze the conversion of L-malate to oxaloacetate. Despite similar enzymatic function, there is little homology at the amino acid level between the two isoforms (approximately 20% sequence identity and approximately 35% sequence homology, see Joh T et al., J Biol Chem, 1987, 262:15127-15131). While the biochemical role of mMDH as an important metabolic enzyme is well documented, the functional significance of cMDH remains unclear. The classical theory of cMDH function is that it helps maintain steady state levels of reducing equivalents in cytoplasm by converting between malate + NAD and oxaloacetate + NADH.

Tryptic digestion of the p36 generated peptides having sequences that matched the sequences of rat, mouse, pig and human cytosolic malate dehydrogenase (cMDH). Both the cMDH substrate L-malate, and an antibody generated against cMDH block cMDH activity. When either of these two molecules was added to reconstituted p36 and pure p45, activity of the nucleic acid channel was blocked, confirming the presence of the cMDH and its specificity in regulating nucleic acid transport across the membrane. Oxaloacetate was also found to inhibit channel activity. These results were surprising in that cMDH has previously been described as a globular cytosol-soluble protein and has not been considered to be associated with cell membranes. Immunostaining of a cell line derived from porcine kidney proximal tubules revealed staining on the margin of cells, confirming that cMDH was located in the cell membranes.

Modulation of Nucleic Acid Transport Channel Protein Complex Activity

The present invention provides a means of identifying compounds that modulate nucleic acid transport activity across cell membranes by detecting activity of the nucleic acid transport channel in the presence of test compounds.

Screening Assays The present invention provides various screening assays for identifying nucleic acid transport channel protein complex modulators, i.e., inhibitors or agonists, useful as targets for diagnosis and/or treatment of conditions arising from accumulation of, or lack of, certain nucleic acids.

The term "nucleic acid transport channel protein complex modulator" is used herein to refer to a compound that can modulate or modify a nucleic acid transport channel protein complex from exhibiting its normal gating activity. Such a modulator may directly affect nucleic acid transport channel protein complex function, substrate recognition by the individual protein subunits, activation, multimerization, or channel formation. A modulator may be a compound that can induce the nucleic acid transport channel protein complex to maintain the channel in an open position. Alternatively, a modulator may refer to a compound that can induce the nucleic acid transport channel protein complex to maintain the channel in a closed position.

"Screening" refers to a process of testing one or a plurality of compounds (including a library of compounds) for some activity. A "screen" is a test system for screening. Screens can be primary, i.e., an initial selection process, or secondary, e.g., to confirm that a compound selected in a primary screen (such as a binding assay) functions as desired (such as in a signal transduction assay). Screening permits the more rapid elimination of irrelevant or non-functional compounds, and thus selection of more relevant compounds for further testing and development. "High throughput screening" involves the automation and robotization of screening systems to rapidly screen a large number of compounds for a desired activity.

The screening assays of the invention are particularly advantageous by permitting rapid evaluation of cellular response. Biological assays, which depend on cell growth, survival, or some other response require substantial amounts of time and resources to evaluate. By detecting individual signals in the expression of the nucleic acid membrane transport channel protein complex, the present invention bypasses tedious and time consuming biological assays.

The present invention contemplates screens for small molecule compounds, including ligand analogs and mimics, as well as screens for natural compounds that bind to and agonize or antagonize nucleic acid transport channel protein complex activity in vivo. Such agonists or antagonists may, for example, interfere in the full activation of nucleic acid transport channel protein complex, that is the channel may not fully assemble or may be assembled defectively because one or more of the monomers does not form the heteromultimer to form a channel; or the pore forms successfully, but remains closed or open. As used herein, the term "compound" refers to any molecule or complex of more than one molecule that affects nucleic acid transport channel protein complex activity. The present invention contemplates screens for synthetic small molecule agents, chemical compounds, chemical complexes, and salts thereof as well as screens for natural products, such as plant extracts or materials obtained from fermentation broths. Other molecules that can be identified using the screens of the invention include proteins and peptide fragments, peptides, nucleic acids and oligonucleotides (particularly triple-helix-forming oligonucleotides), carbohydrates, phospholipids and other lipid derivatives, steroids and steroid derivatives, prostaglandins and related arachadonic acid derivatives, etc.

Natural products libraries can be screened using assays of the invention for such molecules. In another aspect, synthetic combinatorial libraries (Needels et al., Proc Natl Acad Sci USA, 1993, 90:10700-4; Ohlmeyer et al., Proc Natl Acad Sci USA, 1993, 90: 10922-10926; Lam et al., International Patent Publication No. WO 92/00252; Kocis et al, International Patent Publication No. WO 94/28028) and the like can be used to screen for compounds according to the present invention.

Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., Tib Tech, 1996, 14:60).

Contemplated within the scope of the invention is the use of the nucleic acid transport channel protein complex to find modulators of nucleic acid transport channel protein complex activity. Modulating nucleic acid transport channel protein complex activity provides a means of preventing, reducing or ameliorating pathological conditions in which nucleic acid transport channel protein complexes are present. Nucleic acid transport channel protein complex activity may be modulated by compounds or molecules that increase or reduce complex activity, or are antagonists or agonists of the component subunits of the complex, either the channel forming protein or the regulatory protein. Modulators can include molecules containing sulfate, for example, heparin sulfate, which applicants have found blocks nucleic acid channel activity. Other modulators include antibodies to the p45 which block channel activity.

In a specific embodiment, screening assays explore the ability of test compounds that are antagonists or agonists of the regulatory protein subunit, in particular, inhibitors of cMDH, to block transport of nucleic acids through the membrane channel. Such inhibitors include cMDH substrates, such as L-malate, anti-cMDH antibodies, oxaloacetate and the like.

Assays can also explore the ability to block or reduce transport of nucleic acids through the membrane by agents that bind to oligonucleotides.

The screening assays utilize methods known to those of skill in the art as a means of determining channel activity, and as described below.

In vitro cell membrane nucleic acid transport channel protein complex function

Electrophysiology The difference in electric potential between the inside and outside of a cell due to ion movement into or out of the cell can be used to evaluate the permeability of a cell membrane. In vitro, the activity of the large-pore forming proteins are evaluated using the planar lipid bilayer model to detect differences in electric potential on either side of the bilayer (see, e.g., Hanss B et al. 1998). Putative nucleic acid transport channel protein subunits and complexes are placed in proteoliposomes and fused to planar lipid bilayers. The nucleic acid transport channel protein complexes are tested for their ability to form nucleic acid gated channels in the membrane and to determine their activity, that is determine gating kinetics and substrate suitability. Current-voltage relationships are evaluated; the effects of different ion concentrations on either side of the membrane on channel opening or closing may be determined. The effect of various compounds on channel forming activity of the protein complex is evaluated by detecting changes in channel conductance and kinetics. Channel blockade can be tested using compounds known to alter channel activity, for example, using compounds that bind to nucleic acids. Oligonucleotides are known to bind to heparin binding proteins and heparin can block oligonucleotide uptake in some cells. For example, heparan sulfate, a large polyanion similar in molecular weight and valence to unmodified oligonucleotides with phosphodiester backbones, is shown to reduce the probability of the nucleic acid channel to be open.

Transmembrane transport of oligonucleotides Oligonucleotide transport across the channel can be directly measured by radiolabeling oligonucleotides and monitoring their movement across the membrane. ODNs can also be biotinylated, fluorescently labelled or labelled or tagged using methods known in the art.

Applications The nucleic acid transport channel protein complex of the present invention can be used to facilitate the transport of nucleic acids into cells for a number of therapeutic applications. Genes can be transferred to cells using plasmid DNA, protein synthesis can be interrupted with ribozymes and antisense oligodeoxynucleotides, and, genetic defects caused by point mutations can be corrected with RNA/DNA hybrid oligonucleotides (Cole-Strauss et al, Science, 1996, 273:1386-1389). These approaches require intemalization of nucleic acid and trafficking to appropriate intracellular locations.

Intracellular toxicity results primarily from non-sequence specific hybridization of an oligonucleotide with mRNA and or proteins. This can result in a decrease in either a subpopulation of messages or in more global decreases in mRNA levels, see, e.g., Wolf et al. (Proc Natl Acad Sci, 1992, 89: 7305-7309). These authors showed that even specific ODNs with varying degrees of base mismatch will still hybridize with its target. Thus the present invention can be used to transport ODNs into a cell wherein the ODNs will hybridize with a message even if not fully complementary to it and result in down regulation of protein synthesis. Oligonucleotides also interact directly with proteins. For example, S-ODNs bind and inhibit the activity of a variety of kinases (Teasdale et al., Antisense Res Dev, 1994, 4: 295-297; Bergen et al., Antisense Res Dev, 1995, 5:33-38); block the activity of thrombin (Block et al., Nature, 1992, 355:564-566); and S-ODNs inhibit protein kinase C (Stein et al., Biochemistry, 1993, 32: 4855-4861).

In frog oocytes, Cazenave et al. (Nucleic acid research, 1989, 17: 425-4273) observed a global decrease in protein synthesis following administration of S-ODNs. The mechanism is not clear but thought to be due to ODN-protein interaction, blocking protein synthesis machinery. The present invention can be used to reduce nucleic acid toxicity in cells by blocking or reducing channel activity to block or reduce transport of nucleic acids into cells. The kidney is one example of a target organ for modulating nucleic acid transport into cells. There are a number of diseases of the kidney that are thought to be monogenic and, as such, are ideal targets for molecular therapy, for example, Liddle's syndrome, polycystic kidney disease, Wilm's tumor, and some forms of renal cell carcinoma.

The invention can be better understood by reference to the following Examples which are provided by way of illustration and not by way of limitation.

EXAMPLE

A. Purification of the Nucleic Acid Channel Protein A nucleic acid channel protein complex was isolated from renal brush border membranes as described (Rappaport et al., 1995). Briefly, male Sprague-Dawley rats were anesthetized with Inactin and both kidneys were removed. Outer cortical tissue was harvested and cell membranes were separated from other cell components by differential centrifugation, followed by density gradient centrifugation through Percoll gradients as described previously (Leal-Pinto et al., Bioquimica Acta Cicienitica Venezuela, 1987, 38: 157-163). The rat- kidney brush border membranes were solubilized in 1 % CHAPS. SDS-PAGE of an aliquot of this preparation is shown in Figure 1 A. The solubilized membrane preparation was applied sequentially to two ion-exchange columns, (1) HiLoad 16/10 Q Sepharose HP and (2) Mono Q HR 5/5 (Pharmacia Biotech). Fractions of eluate which contained protein as detected by UV spectroscopy were assessed for channel activity in the lipid bilayer system, described below. Active fractions were pooled and analysis by SDS-PAGE revealed the presence of approximately 8 proteins (Figure IB). Pooled active fractions were applied to a hydrophobic interaction column (Source 15 PHE PE 4.6/100, Pharmacia Biotech). The fractions containing the active peak from this separation contained two protein bands; a 45-kDa band (p45) and a second band at 36-kDa (p36) (Figure 1C). Gel electrophoresis in two dimensions indicated that each band contained only a single protein. The active fractions were pooled, 1 OmM DTT was added to the pooled fractions, and the sample was applied to a gel filtration column (Sephadex 75 HR 10/30, Pharmacia Biotech). This chromatography step resulted in two protein peaks as detected by UV spectroscopy. SDS-PAGE revealed that these peaks corresponded to pure p45 (Figure ID) and pure p36. The yield of p36 from this step was low and was at the lower limit of detection on silver stained gels. Nucleic acid dependent channel activity was lost after this final step of purification.

B. Lipid Bilayer Model -Testing Functional Reconstitution of Nucleic Acid Channel Complex and Subunits

The following experiments were performed to determine whether the nucleic acid channel was a heteromultimer of p45 and p36. Proteoliposomes containing either pure p36 or pure p45 were prepared and channel activity was assessed in planar lipid bilayers

Proteoliposomes were prepared by sonicating (80 kHz for 1 minute) purified protein (p36 or p45) with a 1 : 1 mixture of bovine brain phosphatidylethanolamine (10 mg/ml) and phosphatidylserine (10 mg/ml, Avanti Polar Lipids). A lipid bilayer was formed by "painting" a 50-100 A hole with a 1 :1 mixture of the same lipids and to form a high resistance seal between two cups. Each cup was filled with 1 ml of a buffered solution containing 200 mM CsCl, 1 mM CaCl, and 20 mM Hepes, pH 7.4. ODN (20-mer homomultimer of deoxythymidine (Oswel DNA services) was added to a concentration of 5 μM. The cups were connected to a patch clamp amplifier through a head stage with a 10-gigohm feedback resistor and frequency bandwidth of 10 kHz. The cis chamber was defined as the cup connected to the voltage-holding electrode and all voltages were referenced to the trans (ground) side. Stability of the bilayer was determined by clamping voltage between -100 mV and +100 mV. Resistance of at least 100 gigohms and noise of less than 0.2 pA was maintained throughout this range of holding potential. Experiments were initiated by adding proteoliposomes to the trans chamber and confirming stability of the bilayer. Current output of the patch clamp was filtered at 1 kHz through an eight-pole filter, digitized at 0.05 or 0.25 ms/point, and analyzed with commercial software (Pclamp, Version 6.02, Axon Instruments). Channel events with an open time greater than 2.0 ms and a noise level at the open state of less than 2 times background noise were analyzed. Current traces were recorded for each experiment. Membrane holding potential is indicated for each trace and the solid horizontal line indicates zero current. When the current trace overlies this line the channel was in the closed state. p45 Typical channel activity of the isolated nucleic acid channel complex

(Hanss et al. 1998) is illustrated in Figure 2A. Figure 2B shows a current output trace from proteoliposomes formed with a fraction containing pure p45; channel activity was seen in the absence of ODN. Channel conductance was -60 pS, a value 6 fold higher than the reported conductance of the nucleic acid dependent activity of the channel complex. Ion substitution studies indicated that pure p45 alone was non-selective, conducting both cations and anions equally and open probability approached 1.00 with only occasional transitions to zero or a higher level of current. The channel activity of p45 alone was blocked by heparan sulfate at a concentration previously shown to block nucleic acid dependent channel complex activity (Hanss et al., 1998). Addition of ODN resulted in modulation of p45-alone activity but did not significantly change the current (Figure 2C). These data indicated that pure p45 formed a pore or channel in the membrane that was not dependent on ODN for gating, and conducted ions other than ODN. p36 Channel activity was not observed when pure p36 was reconstituted in either the absence (Figure 2D) or presence (Figure 2E) of ODN, indicating that p36 alone did not form a channel. When p45-containing proteoliposomes and p36-containing proteoliposomes were simultaneously fused to the model membrane, ODN-independent channel activity was not observed. The addition of 10 μM ODN resulted in channel activity (Figure 2F) comparable to that observed for the isolated affinity purified channel complex (Figure 2A), i.e., channel conductance was 16 pS and gating kinetics were similar to previous reports (Hanss et al., 1998). Ion substitution studies indicated the channel was selective for nucleic acids. The data confirmed that the nucleic acid transport channel protein complex was a heteromul timer of p45 and p36. p45 formed a non-selective transmembrane pore. The p36 subunit had no channel activity when reconstituted alone. When added to the p45 subunit, the p36 interacted with the p45 to form a nucleic acid-gated, nucleic acid-conducting channel complex.

C. Amino Acid Sequencing

FPLC purified p45 and p36 were digested and sequenced by Tandem Mass Spectrometry using standard techniques. Four peptide sequences from p45 (SEQ ID NOs:l- 4) and four peptides from p36 were identified. Peptides generated from the digest of p36, VIVVGNPANTNCLTASK (SEQ ID NO:6), ENFACLTR (SEQ ID NO: 7), LGVTADDVK (SEQ ID NO: 8), NVIIWGNHSSTQYPDVN (SEQ ID NO:9), GEFITTVQQR (SEQ ID NO: 10) and FVEGLPINDFSREK (SEQ ID NO: 11) were identical to rat, mouse, pig, and human cMDH. Alignment of the p36 peptides with the sequence of human cMDH (SEQ ID NO: 5) is shown in Figure 3. A hydropathy plot (Kyte-Doolittle) of human cMDH is shown in Figure 4 A. The first 80 amino acids of human cMDH contain several hydrophobic domains as indicated by hydrophobicity scores above zero. Prediction of transmembrane spanning domains in human cMDH using TMpred algorithms is shown in Figure 4B. Transmembrane helix scores greater than 500 indicated, with high probability, transmembrane spanning domains. Two potential membrane-spanning domains were predicted within the first 50 amino acids of human cMDH.

D. Blockade of the Nucleic Acid Channel by L-malate

The following experiments were performed to confirm that cMDH was the p36 subunit of the nucleic acid transport channel protein complex. L-malate, a substrate for cMDH, was tested to see if by binding to cMDH, channel function was altered. In these experiments proteoliposomes were prepared as described above using both p45 and p36. The proteoliposomes were added to the bilayer chamber and allowed to fuse with the membrane. Varying concentrations (5, 20 and 75 μM) of L-malate were added to the bilayer chambers and the current output was measured and compared to a control (no L-malate added). Current output traces are shown in Figure 5. Reconstitution of the channel complex resulted in channel activity, indicated by clear transitions between the open and closed states (Figure 5A); open probability was 0.36. Addition of 5 μM L-malate to both solution chambers resulted in a decrease in channel open probability to 0.10 (Figure 5B). Increasing L-malate concentration to 75 μM resulted in complete blockade of channel activity (Figure 5C). Channel activity was restored when L- malate was washed from the solution chambers. In four additional experiments, 20 μM L- malate resulted in a 79+11% decrease in channel open probability. The inhibition of channel activity by L-malate indicated that the p36 cMDH was the regulatory subunit of the channel.

E. Analysis using cMDH Antiserum Since an antibody against cMDH was not available commercially, we generated a polyclonal antiserum against pig heart cMDH (Sigma Chemical Co.) in mice using methods known in the art. The results of these experiments are shown in Figure 6. ELIS A (Lauritzen et al., Antibody Techniques (Malic VS and Lillehoj EP, eds) pp 27-258, Academic Press, New York, 1994) results plotted in Figure 6 A show that the mouse immunized with cMDH mounted a strong immune response against cMDH as indicated by a titer of greater than 1 : 10,000.

Specificity of antiserum for cMDH Serum was tested for specificity in western blots of either pig-heart cMDH (Sigma Chemical Co.) or whole kidney lysate from rat. In western blots, this serum recognized purified pig-heart cMDH (Figure 6B) and was immunoreactive with only a single protein that co-migrated with a cMDH control when blots of whole kidney lysate were probed (Figure 6C). cMDH antiserum blocks activity of the nucleic acid channel Antiserum at a dilution of 1 :200 resulted in an 80+13% decrease in channel open probability. Figures 6D and 6E show the results of an experiment in which antiserum was tested for interaction with the channel complex. Anti-cMDH antiserum resulted in a significant reduction in the nucleic acid channel open probability from a control of 0.11 (Figure 6D) to 0.4 after addition of 5 μl of serum (1 :200, Figure 6E) indicating that the cMDH specific antiserum blocked the channel. Normal mouse serum was without effect.

Immunolocalization ofcMDH in membranes of cultured cells LLC-PK cells derived from pig kidney proximal tubules were grown to confluence on coverslips, immunostained with anti-cMDH antiserum, and examined by laser scanning confocal microscopy. Confocal laser scanning microscopy was performed at the Mt. Sinai School of Medicine Confocal Scanning Laser Microscopy core facility, supported with funding from NIH shared instrumentation grant (1 S10 RRO 9145-01) and NSF Major Research Instrumentation grant (DBI-9724504). Anti-cMDH antiserum was immunoreactive with cell membranes confirming cell membrane localization of cMDH. Diffuse cytoplasm and perinuclear immunostaining was also present suggesting localization to these regions as well. Pre-immune sera were not immunoreactive with LLC-PK cells. Immunoreactivity along the margin of cells was consistent with localization of cMDH with the cell membrane. Immunolocalization of cMDH in the membranes concurred with the computational analyses (Figure 3C) of cMDH, confirming that cMDH associated with p45 at the cell membrane to regulate channel activity.

F. Functional Reconstitution of Nucleic Acid Channel Activity with cMDH

To confirm that cytosolic malate dehydrogenase is the regulatory subunit of the nucleic acid channel, proteoliposomes were formed with either pure p45 or pig-heart cMDH (Sigma Chemical Co.), fused with lipid bilayers either individually or together, and channel activity characterized. Proteoliposomes made with pure p45 demonstrated nucleic acid independent channel activity (Figure 7 A) similar to that described in Figure 5. Proteoliposomes containing pig-heart cMDH did not show channel activity (Figure 7B). Channel activity was not seen when p45 and commercial cMDH were reconstituted together in the absence of ODN (Figure 7C); however, when 10 μM ODN was added, channel gating was observed (Figure 7D). Channel conductance of reconstituted p45 and pig-heart cMDH was 12.4+3.9 pS. These data provided direct evidence that cytosolic malate dehydrogenase was the regulatory subunit of the nucleic acid channel.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Various patents, patent applications, and publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

What is claimed is: L A cell membrane nucleic acid channel transport protein complex comprising a first subunit, wherein said first subunit is a cell membrane channel forming protein having a molecular weight of about 45kDa and having the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; and a second subunit wherein the second subunit is a regulatory protein that regulates the specificity of the channel forming protein for nucleic acid transport.

2. The method of claim 1 , wherein the regulatory protein is cytosolic malate dehydrogenase.

3. The complex of claim 2, wherein the regulatory protein has an amino acid sequence of SEQ ID NO: 5.

4. The complex of claim 1, located in a cell membrane, wherein the cell membrane is not a kidney cell membrane.

5. The complex of claim 4, wherein the cell membrane is part of a whole intact cell.

6. A method of regulating the transport of nucleic acids into a cell, which method comprises modifying the activity of a cell membrane channel forming protein having the amino acid sequences of SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 to have nucleic acid specific transport activity.

7. The method of claim 6, wherein the cell is modified to express a second protein that regulates the specificity of the cell surface channel forming protein for transporting nucleic acids.

8. The method of claim 7, wherein the second protein is cytosolic malate dehydrogenase.

9. The method of claim 8, wherein the second protein has an amino acid sequence of SEQ ID NO: 5.

10. A method of producing a cell having a nucleic acid transport channel protein complex, which method comprises introducing into a cell membrane a cell membrane channel forming protein having the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 and a protein that regulates the specificity of the cell surface channel forming protein for transporting nucleic acids.

1 1. The method of claim 10, wherein the protein that regulates the specificity of the cell surface channel forming protein for nucleic acids is cytosolic malate dehydrogenase.

12. The method of claim 11 , wherein the protein that regulates the specificity of the cell surface channel forming protein for nucleic acids has the amino acid sequence of SEQ ID NO: 5.

13. A method of identifying compounds which modulate the activity of a cell membrane nucleic acid transport channel protein complex, which method comprises detecting the activity of the nucleic acid transport channel protein complex in the presence of a test compound.

14. The method of claim 13, wherein the compound is an agonist or antagonist of channel protein complex activity.

15. The method of claim 13, wherein the compound blocks the activity of cytosolic malate dehydrogenate.

16. The method of claim 15, wherein the compound is L-malate or oxaloacetate.

17. A method ofreducing nucleic acid toxicity in kidney cells, which method comprises administering an agent that inhibits or reduces the activity of a nucleic acid transport channel protein complex wherein the transport of nucleic acids into the cell is reduced or inhibited.

18. The method of claim 17, wherein the agent blocks cytosolic malate dehydrogenase activity.

19. The method of claim 18, wherein the compound is L-malate or oxaloacetate.