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Title:  Human voltage gated sodium channel .beta.1A subunit and methods of use
United States Patent: 
7,063,953
Issued: 
June 20, 2006
Inventors:  Qin; Ning (Blue Bell, PA); Codd; Ellen (Blue Bell, PA); D'Andrea; Michael (Cherry Hill, NJ)
Assignee:
  Ortho-McNeil Pharmaceutical, Inc. (Raritan, NJ)
Appl. No.:  401916
Filed: 
March 28, 2003


 

George Washington University's Healthcare MBA


Abstract

DNAs encoding human voltage gated sodium channel .beta.1A subunit have been cloned and characterized. The recombinant protein is capable of forming biologically active protein. The cDNA's have been expressed in recombinant host cells that produce active recombinant protein. The recombinant protein is also purified from the recombinant host cells. In addition, the recombinant host cells are utilized to establish a method for identifying modulators of the receptor activity, and receptor modulators are identified.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of Human Voltage Gated Sodium Channel .beta.1A Subunit Nucleic Acid

The voltage gated sodium channel is a multi-subunit protein complex containing a pore forming subunit .alpha., and two regulatory subunits .beta.1 and .beta.2. While the .alpha. subunit determines the basic properties of the channel .beta.1 and .beta.2 subunits modulate almost all aspects of the channel properties including voltage dependent gating, voltage dependent activation and inactivation, and most strikingly, increasing functional channel density on the membrane. From molecular pharmacologic and electrophysiologic perspectives, there are more subtypes of voltage gated sodium channel in the excitable cells than cloned .alpha. subunits. This may be partially due to the existence of more .alpha. subunits in nature to be cloned and characterized. On the other hand, one might also expect that there might be more than one type of .beta.1 and .beta.2 subunits. In other words, the variety of voltage gated sodium channel may result from the different types of .alpha. subunit associating with one type of .beta.1 and .beta.2 subunits, and vice versa. In fact, biochemical study had revealed that there are more than one type of sodium channel .beta.1 subunit as determined by Western blot with .beta.1 specific antibody (Sutkowski, et al. 1990).

Recently .beta.1A, a novel voltage-gated sodium channel subunit and splice variant of .beta.1, has been cloned and was reported to increase sodium current density at the plasma membrane and change voltage dependent kinetics when co-expressed with .alpha.IIA subunits in CHL fibroblasts (Kazan-Gillespie et al., 2000). .beta.1A is developmentally regulated in the brain. The subcellular distribution studies of rat VGSC .beta.1A subunit demonstrated that it was altered in addition to being up-regulated in the DRG neurons as a consequence of peripheral nerve injury. Furthermore, rat VGSC .beta.1A subunit may be important in the regulation of the aberrant array of sodium channels expressed subsequent to nerve injury.

To study the human .beta.1A subunit, we first tried to clone the subunit using a homologous cloning strategy. Since .beta.1A is an intron retained splicing variant at the carboxyl terminus of the .beta.1 subunit, the forward primer was designed based on the human VGSC .beta.1 subunit; while the reverse primer was designed based on rat VGSC .beta.1A subunit. Unexpectedly, this pair of primers failed to amplify any DNA fragment from human adrenal gland, fetal brain and adult brain Marathon.TM. ready cDNA libraries. Three different reverse primers based on the sequence encoding the carboxyl terminus of rat .beta.1A subunit were then designed. However, none of these primers paired with forward primer could amplify any DNA fragment from the above cDNA libraries under several PCR conditions, suggesting that human VGSC .beta.1A subunit was significantly different from its counterpart in rat.

In order to clone the splicing variant of human VGSC .beta.1A subunit, a Rapid Amplification of cDNA End (RACE-PCR) technique was used. Unlike regular RT-PCR that requires two gene specific primers, RACE-PCR requires only one specific primer pairing with a universal primer (AP1 or AP2) for RT-PCR amplification. This requires adding an adaptor recognized by the universal primer to the end of each cDNA when the library was made. Currently, this type of cDNA library is commercially available (Marathon.TM. ready cDNA library, Clontech). Therefore, by this technique, human VGSC .beta.1A subunit could be amplified with a human .beta.1 specific primer (SB1-10, see example 1) without knowing the sequence of human .beta.1A carboxyl terminus. The technical difficulty of this application is to effectively distinguish novel .beta.1A from the .beta.1 subunit because of using .beta.1 subunit specific primer for RACE-PCR. To solve this problem, the transformants were pre-screened by PCR with a pair of primers recognizing only the human VGSC .beta.1 subunit and the negative clones (which could not be amplified by such PCR) would be subjected for further characterization and sequencing. With this strategy, a novel human VGSC .beta.1A subunit was cloned from human adrenal gland Marathon.TM. ready cDNA library, and subsequently amplified from human fetal brain Marathon.TM. ready cDNA library with human .beta.1A specific primers.

Analysis of the primary sequence revealed that the human VGSC .beta.1A subunit is also a splicing variant of the .beta.1 subunit with a retained intron and in frame stop codon. This novel VGSC .beta. subunit also contains the basic structure of VGSC .beta. subunit: an amino-terminal extracellular immunoglobulin-like motif and a carboxyl-terminal transmembrane domain. However, the human VGSC .beta.1A subunit is significantly different from its rat counterpart. They share only about 35% identity at their carboxyl terminal coding region.

The present invention relates to DNA encoding human VGSC .beta.1A subunit that was isolated from human VGSC .beta.1A subunit producing cells. The term "Human VGSC .beta.1A subunit", as used herein, refers to protein which can specifically function as a channel subunit. That is, it can combine with the other protein subunits to form a functioning calcium channel.

The recombinant protein is useful to identify modulators of the functional human VGSC .beta.1A subunit. Northern blot analysis demonstrated that the VGSC .beta.1A subunit was widely distributed in a variety tissues including, but not limited, in brain, heart, skelet al muscle, liver, lung, placenta, kidney and pancreas. In brain, the VGSC .beta.1A subunit expresses most highly in the cerebellum region. Immunohistochemical study also demonstrates that the VGSC .beta.1A was not only expressed in dorsal root ganglia (DRG), but is also up-regulated after nerve injury, suggesting it plays a role in neuropathic pain. Alteration in sodium channel expression and/or function can have a profound influence on the firing properties of peripheral and central neurons, and many other tissues. Modulators of VGSC .beta.1A can be identified in the assays of this invention and tested for their use as therapeutic agents for neuropathic pain, chronic pain, febrile seizures and general epilepsy, local anesthetics, antiarrhythmics and anticonvulsants as well as many other human diseases related to sodium channels (Wallace, et al. 1998, Porreca, et al, 1999, Balser 1999). Human VGSC .beta.1A may also be useful in human diseases where other .beta.1 sodium channel alterations are linked to aberrant sodium channel activity, such as generalized epilepsy with febrile seizures plus (GEFS+) and congenital long-QT syndrome (LQT) (a cardiac arrhythmia characterized in part by prolonged ventricular repolarization).

Moreover, as provided in Example 12 below, the expression of the .beta.1A subunit and .beta.1 was investigated in DRG neurons from a nerve ligation model of neuropathic pain in rats using immunohistochemistry and image analysis. The levels of .beta.1A subunit and .beta.1 expression were increased most notably in the nociceptive DRG neurons, although there were increases in the sensory DRG neurons as well. However, the subcellular labeling of these two polypeptides differed dramatically in the DRG neurons subsequent to peripheral nerve injury. These studies demonstrated that peripheral nerve injury is associated with upregulation of .beta.1A subunit and .beta.1 sodium channel subunits and altered cellular distribution patterns of .beta.1A in DRG. This is evidence that the sodium channel .beta.1A subunit is important in the regulation of the aberrant sodium channel expression in DRG neurons subsequent to nerve injury.

Thus, this invention also contemplates the use of screening assays employing the human .beta.1A subunit to identify modulators capable of binding to the .beta.1A subunit. The modulators that are capable of binding to the human .beta.1A subunit can then be used as a therapeutic, such as in a pharmaceutical composition, for the treatment of or as a method for decreasing neuropathic pain in a human. The pharmaceutical compositions comprising the modulator of the human .beta.1A subunit are delivered to cells binding to a sodium channel .beta.1A subunit-expressing cell in the human and function to decrease neuropathic pain.

A variety of cells and cell lines may be used to isolate Human .beta.1A sodium channel subunit cDNA using primers selected based on the nucleotide sequence encoding the human .beta.1A sodium channel subunit.

Any of a variety of procedures known in the art may be used to molecularly clone Human .beta.1A sodium channel subunit DNA to obtain related sequences or allelic variants. These methods include, but are not limited to, direct functional expression of the Human .beta.1A sodium channel subunit genes following the construction of a Human .beta.1A sodium channel subunit-containing cDNA library in an appropriate expression vector system. Another method is to screen Human .beta.1A sodium channel subunit-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled oligonucleotide probe designed from the amino acid sequence of the Human .beta.1A sodium channel subunit subunits. An additional method consists of screening a Human .beta.1A sodium channel subunit-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the Human .beta.1A sodium channel subunit protein. This partial cDNA is obtained by the specific PCR amplification of Human .beta.1A sodium channel subunit DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified Human .beta.1A sodium channel subunit protein.

Another method is to isolate RNA from human VGSC .beta.1A subunit-producing cells and translate the RNA into protein via an in vitro or an in vivo translation system. The translation of the RNA into a peptide a protein will result in the production of at least a portion of the Human .beta.1A sodium channel subunit protein which can be identified by, for example, immunological reactivity with an anti-human .beta.1A sodium channel subunit antibody or by biological activity of Human .beta.1A sodium channel subunit protein. In this method, pools of RNA isolated from Human .beta.1A sodium channel subunit-producing cells can be analyzed for the presence of an RNA that encodes at least a portion of the Human .beta.1A sodium channel subunit protein. Further fractionation of the RNA pool results in purification of the Human .beta.1A sodium channel subunit RNA from non-Human .beta.1A sodium channel subunit RNA. The peptide or protein produced by this method may be analyzed to provide amino acid sequences which in turn are used to provide primers for production of Human .beta.1A sodium channel subunit cDNA, or the RNA used for translation can be analyzed to provide nucleotide sequences encoding Human .beta.1A sodium channel subunit and produce probes for this production of Human .beta.1A sodium channel subunit cDNA. This method is known in the art and can be found in, for example, Maniatis, T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.

It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have Human .beta.1A sodium channel subunit activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate Human .beta.1A sodium channel subunit cDNA may be done by first measuring cell associated Human .beta.1A sodium channel subunit activity using the measurement of Human .beta.1A sodium channel subunit-associated biological activity or a ligand binding assay.

Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

It is also readily apparent to those skilled in the art that DNA encoding Human .beta.1A sodium channel subunit may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

In order to clone the human sodium channel .beta.1A subunit gene by the above methods, the amino acid sequence of Human sodium channel .beta.1A subunit may be necessary. To accomplish this, Human .beta.1A sodium channel subunit protein may be purified and partial amino acid sequence determined by automated sequencers. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from the protein is determined for the production of primers for PCR amplification of a partial Human .beta.1A sodium channel subunit DNA fragment.

Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the Human .beta.1A sodium channel subunit sequence but will be capable of hybridizing to Human .beta.1A sodium channel subunit DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still sufficiently hybridize to the Human sodium channel .beta.1A subunit DNA to permit identification and isolation of Human sodium channel .beta.1A subunit encoding DNA. DNA isolated by these methods can be used to screen DNA libraries from a variety of cell types, from invertebrate and vertebrate sources, and to isolate homologous genes.

Purified biologically active Human sodium channel .beta.1A subunit may have several different physical forms. The Human sodium channel .beta.1A subunit may exist as a full-length nascent or unprocessed polypeptide, or as partially processed polypeptides or combinations of processed polypeptides. The full-length nascent Human .beta.1A sodium channel subunit polypeptide may be post-translationally modified by specific proteolytic cleavage events that results in the formation of fragments of the full length nascent polypeptide. A fragment or physical association of fragments may have the full biological activity associated with Human .beta.1A sodium channel subunit however, the degree of Human .beta.1A sodium channel subunit activity may vary between individual Human .beta.1A sodium channel subunit fragments and physically associated Human .beta.1A sodium channel subunit polypeptide fragments.

Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the Human .beta.1A sodium channel subunit sequence but will be capable of hybridizing to Human .beta.1A sodium channel subunit DNA even in the presence of DNA oligonucleotides with mismatches under appropriate conditions. Under alternate conditions, the mismatched DNA oligonucleotides may still hybridize to the Human .beta.1A sodium channel subunit DNA to permit identification and isolation of Human .beta.1A sodium channel subunit encoding DNA.

DNA encoding Human .beta.1A sodium channel subunit from a particular organism may be used to isolate and purify homologues of Human .beta.1A sodium channel subunit from other organisms. To accomplish this, the first Human .beta.1A sodium channel subunit DNA may be mixed with a sample containing DNA encoding homologues of Human .beta.1A sodium channel subunit under appropriate hybridization conditions. The hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified therefrom.

Functional Derivatives/Variants

It is known that there is a substantial amount of redundancy in the various codons that code for specific amino acids. Therefore, this invention is also directed to those DNA sequences that contain alternative codons that code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein, which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of aliphatic amino acids Alanine, Valine, Leucine and Isoleucine; interchange of the hydroxyl residues Serine and Threonine, exchange of the acidic residues Aspartic acid and Glutamic acid, substitution between the amide residues Asparagine and Glutamine, exchange of the basic residues Lysine and Arginine and human .beta.1A sodium channel subunits among the aromatic residues Phenylalanine, Tyrosine may not cause a change in functionality of the polypeptide. Such substitutions are well known and are described, for instance in Molecular Biology of the Gene, 4.sup.th Ed. Bengamin Cummings Pub. Co. by Watson et al.

It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis, chimeric substitution, and gene fusions. Site-directed mutagenesis is used to change one or more DNA residues that may result in a silent mutation, a conservative mutation, or a nonconservative mutation. Chimeric genes are prepared by swapping domains of similar or different genes to replace similar domains in the human .beta.1A sodium channel subunit gene. Similarly, fusion genes may be prepared that add domains to the human .beta.1A sodium channel subunit gene, such as an affinity tag to facilitate identification and isolation of the gene. Fusion genes may be prepared to replace regions of the human .beta.1A sodium channel subunit gene, for example to create a soluble version of the protein by removing a transmembrane domain or adding a targeting sequence to redirect the normal transport of the protein, or adding new post-translational modification sequences to the human .beta.1A sodium channel subunit gene. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand. All such changes of the polynucleotide or polypeptide sequences are anticipated as useful variants of the present invention so long as the original function of the polynucleotide or polypeptide sequence of the present invention is maintained as described herein.

IDENTITY or SIMILARITY, as known in the art, are relationships between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity and similarity between two polynucleotide or two polypeptide sequences, both terms are well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., (1988) SIAM J. Applied Math., 48, 1073). Methods commonly employed to determine identity or similarity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., (1988) SIAM J. Applied Math., 48, 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., (1984) Nucleic Acids Research 12(1), 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., (1990) J. Molec. Biol. 215, 403).

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single-, and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. Polynucleotides embraces short polynucleotides often referred to as oligonucleotide(s).

The term polypeptides, as used herein, refers to the basic chemical structure of polypeptides that is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.

Among the known modifications which may be present in polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., (1990) Meth. Enzymol. 182, 626 646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", (1992) Ann. N.Y. Acad. Sci. 663, 48 62.

It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli or other cells, prior to proteolytic processing, almost invariably will be N-formylmethionine. During post-translational modification of the peptide, a methionine residue at the NH.sub.2-terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.

The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made by expressing a cloned gene in a host, for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacterial hosts such as, for example, E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized recombinantly by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. A polynucleotide variant is a polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. As noted below, changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Also as noted below, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed above.

A polypeptide variant is a polypeptide that differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. As used herein, a "functional derivative" of Human .beta.1A sodium channel subunit is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of Human .beta.1A sodium channel subunit. The term "functional derivatives" is intended to include the "fragments" "variants" "degenerate variants" "analogs" and "homologues" or to "chemical derivatives" of Human .beta.1A sodium channel subunit. Useful chemical derivatives of polypeptide are well known in the art and include, for example, covalent modification of one or more reactive organic sites contained within the polypeptide with a secondary chemical moiety. Well known cross-linking reagents are useful to react to amino, carboxyl, or aldehyde residues to introduce, for example, an affinity tag such as biotin, a fluorescent dye, or to conjugate the polypeptide to a solid phase surface (for example to create an affinity resin). The term "fragment" is meant to refer to any polypeptide subset of the Human .beta.1A sodium channel subunit.

A molecule is "substantially similar" to a Human .beta.1A sodium channel subunit if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the entire Human .beta.1A sodium channel subunit molecule or to a fragment thereof. Further particularly preferred in this regard are polynucleotides encoding variants, analogs, derivatives and fragments of SEQ.ID.NO.:13, and variants, analogs and derivatives of the fragments, which have the amino acid sequence of the polypeptide of SEQ.ID.NO.:14 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the gene of SEQ.ID.NO.:13. Also especially preferred in this regard are conservative substitutions. Most highly preferred are polynucleotides encoding polypeptides having the amino acid sequence of SEQ.ID.NO.:14, without substitutions.

Further preferred embodiments of the invention are polynucleotides that are at least 75% identical over their entire length to a polynucleotide encoding the polypeptide having the amino acid sequence set out in SEQ.ID.NO.:14, and polynucleotides which are complementary to such polynucleotides. Yet other preferred embodiments of the invention are polynucleotides that are at least 75% identical over a consecutive portion of their length to a polynucleotide encoding the polypeptide having the amino acid sequence 150 to 268 set out in SEQ.ID.NO.:14, and polynucleotides which are complementary to such polynucleotides. Alternatively, highly preferred are polynucleotides that comprise a region that is at least 80% identical, more highly preferred are polynucleotides at comprise a region that is at least 90% identical, and among these preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% identity are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the most preferred. The polynucleotides, which hybridize to be polynucleotides described herein, in a preferred embodiment, encode polypeptides, which retain substantially the same biological function or activity as the polypeptide characterized by the deduced amino acid sequence of SEQ.ID.NO.:14. Preferred embodiments in this respect, moreover, are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ.ID.NO.:13. The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.

There are a large numbers of polynucleotide hybridization techniques known in the art including hybridizations coupling DNA to DNA, RNA to RNA and RNA to DNA. All of these methods can incorporate stringent hybridization conditions to facilitate the accurate identification of nucleic acid targeting to a hybridizable probe. As is known in the art, methods vary depending on the substrate used for hybridization and Maniatis et al. supra, as well as a variety of references in the art detail a number of stringent hybridization techniques. In one example, DNA or RNA samples to be probed are immobilized on a suitable substrate such as nitrocellulose, nylon, polyvinylidene difluoride, or the like. A purified probe, preferably with sufficient specific activity (generally greater than about 10.sup.8 cpm/.mu.g probe), substantially free of contaminating DNA, protein or unincorporated nucleotides is used. Where nitrocellulose is used, and the immobilized nucleic acid is DNA immobilized on nitrocellulose, the nitrocellulose with DNA is incubated with a hybridization solution comprising 50% formamide-deionized, 6.times.SSC, 1% SDS, 0.1% Tween 20 and 100 .mu.g/ml t RNA at 42.degree. C. for 15 minutes. Probe is added and the nitrocellulose is further immobilized at 42.degree. C. for about 12 19 hours. The nitrocellulose is then washed in at least two successive washes at 22.degree. C. followed by stringent washes at 65.degree. C. in a buffer of 0.04M sodium phosphate, pH 7.2, 1% SDS and 1 mM EDTA. Conditions for increasing the stringency of a variety of nucleotide hybridizations are well known in the art.

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding the sequences of SEQ.ID.NO.:13 and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to SEQ.ID.NO.:13. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less. For example, the coding region of the gene of the invention may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA, to determine to which members of the library the probe hybridizes.

The polypeptides of the present invention include the polypeptide of SEQ.ID.NO.:14 (in particular the mature polypeptide) as well as polypeptides which have at least 75% identity to the polypeptide of SEQ.ID.NO.:14, preferably at least 80% identity to the polypeptide of SEQ.ID.NO.:14, and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ.ID.NO.:14 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ.ID.NO.:14 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. Representative examples of polypeptide fragments of the invention, include, for example, truncation polypeptides of SEQ.ID.NO.:14.

Truncation polypeptides include polypeptides having the amino acid sequence of SEQ.ID.NO.:14, or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred in this aspect of the invention are fragments characterized by structural or functional attributes of the polypeptide characterized by the sequences of SEQ.ID.NO.:14. Preferred embodiments of the invention in this regard include fragments that comprise .alpha.-helix and .alpha.-helix forming regions, .beta.-sheet and .beta.-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, .alpha. amphipathic regions, .beta. amphipathic regions, flexible regions, surface-forming regions, substrate binding region, high antigenic index regions of the polypeptide of the invention, and combinations of such fragments. Preferred regions are those that mediate activities of the polypeptides of the invention. Most highly preferred in this regard are fragments that have a chemical, biological or other activity of the response regulator polypeptide of the invention, including those with a similar activity or an improved activity, or with a decreased undesirable activity.

Recombinant Expression of Human .beta.1A Sodium Channel Subunit

The cloned Human .beta.1A sodium channel subunit DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant Human .beta.1A sodium channel subunit protein. Techniques for such manipulations are fully described in Maniatis, T. et al., supra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria including E. coli, bluegreen algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells.

Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria-invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to express recombinant Human .beta.1A sodium channel subunit in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant Human .beta.1A sodium channel subunit expression, include but are not limited to, pMAMneo (Clontech), pIRES vectors (Clontech), pTET-On and pTET-Off (Clontech), pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593, ATCC, Manassas, Va.) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).

A variety of bacterial expression vectors may be used to express recombinant Human .beta.1A sodium channel subunit in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant Human .beta.1A sodium channel subunit expression include, but are not limited to, pET vectors (Novagen), pGEX vectors (Pharmacia) and pQE vectors (Qiagen).

A variety of fungal cell expression vectors may be used to express recombinant Human .beta.1A sodium channel subunit in fungal cells such as yeast. Commercially available fungal cell expression vectors which may be suitable for recombinant Human .beta.1A sodium channel subunit expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).

A variety of insect cell expression vectors may be used to express recombinant Human .beta.1A sodium channel subunit in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of Human .beta.1A sodium channel subunit include, but are not limited to, pBlueBacII (Invitrogen).

DNA encoding Human .beta.1A sodium channel subunit may be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including, but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including, but not limited to, drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include, but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, HEK-293 (ATCC CRL1573), PC12 (ATCC CRL-1721).

The expression vector may be introduced into host cells via any one of a number of techniques including, but not limited to, transformation, transfection, protoplast fusion, lipofection, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce Human sodium channel .beta.1A subunit protein. Identification of Human .beta.1A sodium channel subunit expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-human .beta.1A sodium channel subunit antibodies, and the presence of host cell-associated Human .beta.1A sodium channel subunit activity.

Expression of Human sodium channel .beta.1A subunit DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA or mRNA isolated from Human .beta.1A sodium channel subunit producing cells can be efficiently translated in various cell-free systems including, but not limited to, wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems including, but not limited to, microinjection into frog oocytes, with microinjection into frog oocytes being generally preferred.

To determine the Human .beta.1A sodium channel subunit DNA sequence(s) that yields optimal levels of Human .beta.1A sodium channel subunit activity and/or Human .beta.1A sodium channel subunit protein, Human .beta.1A sodium channel subunit DNA molecules including, but not limited to, the following can be constructed: the full-length open reading frame of the Human .beta.1A sodium channel subunit cDNA encoding the 32 kDa protein from approximately base 4 to approximately base 808 (these numbers correspond to first nucleotide of first methionine and last nucleotide before the first stop codon) and several constructs containing portions of the cDNA encoding Human .beta.1A sodium channel subunit protein. All constructs can be designed to contain none, all or portions of the 5' or the 3' untranslated region of Human .beta.1A sodium channel subunit cDNA. Human .beta.1A sodium channel subunit activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the Human .beta.1A sodium channel subunit DNA cassette yielding optimal expression in transient assays, this Human .beta.1A sodium channel subunit DNA construct is transferred to a variety of expression vectors, for expression in host cells including, but not limited to, mammalian cells, baculovirus-infected insect cells, E. coli, and the yeast S. cerevisiae.

Assay Methods for Human .beta.1A Sodium Channel Subunit

Host cell transfectants and microinjected oocytes may be used to assay both the levels of functional Human .beta.1A sodium channel subunit activity and levels of total Human .beta.1A sodium channel subunit protein by the following methods. In the case of recombinant host cells, this involves the co-transfection of one or possibly two or more plasmids, containing the Human sodium channel .beta.1A subunit DNA encoding one or more fragments or subunits. In the case of oocytes, this involves the co-injection of synthetic RNAs for Human sodium channel .beta.1A subunit protein. Following an appropriate period of time to allow for expression, cellular protein is metabolically labeled with, for example .sup.35S-methionine for 24 hours, after which cell lysates and cell culture supernatants are harvested and subjected to immunoprecipitation with polyclonal antibodies directed against the Human .beta.1A sodium channel subunit protein.

Levels of Human .beta.1A sodium channel subunit protein in host cells are quantitated by immunoaffinity and/or ligand affinity techniques. Cells expressing Human .beta.1A sodium channel subunit can be assayed for the number of Human .beta.1A sodium channel subunit molecules expressed by measuring the amount of radioactive [ligand] binding to cell membranes. Human .beta.1A sodium channel subunit-specific affinity beads or Human .beta.1A sodium channel subunit-specific antibodies are used to isolate for example .sup.35S-methionine labeled or unlabelled Human .beta.1A sodium channel subunit protein. Labeled Human .beta.1A sodium channel subunit protein is analyzed by SDS-PAGE. Unlabelled Human .beta.1A sodium channel subunit protein is detected by Western blotting, ELISA or RIA assays employing Human .beta.1A sodium channel subunit specific antibodies.

Other methods for detecting Human .beta.1A sodium channel subunit activity involve the direct measurement of Human .beta.1A sodium channel subunit activity in, whole cells transfected with Human .beta.1A sodium channel subunit cDNA or oocytes injected with Human .beta.1A sodium channel subunit mRNA and optionally .alpha. sodium channel subunit mRNA. Human .beta.1A sodium channel subunit activity is measured by biological characteristics of the host cells expressing Human .beta.1A sodium channel subunit DNA. In the case of recombinant host cells expressing Human .beta.1A sodium channel subunit patch voltage clamp techniques can be used to measure channel activity and quantify modification of .alpha. sodium channel subunit ion flux as a function of Human .beta.1A sodium channel subunit protein. In the case of oocytes patch clamp as well as two-electrode voltage clamp techniques can be used to measure sodium channel activity and quantify Human .beta.1A sodium channel subunit protein.

Cell Based Assays

The present invention provides a whole cell or isolated cell membrane method to detect compound modulation of human .beta.1A sodium channel subunit. The method comprises the steps;

1) contacting a compound, and a cell or isolated cell membrane that contains functional human .beta.1A sodium channel subunit, and

2) measuring a change in the cell or isolated cell membrane in response to modified human .beta.1A sodium channel subunit function by the compound.

The amount of time necessary for cell or cell membrane contact with the compound is empirically determined, for example, by running a time course with a known human .beta.1A sodium channel subunit modulator and measuring cellular changes as a function of time.

The measurement means of the method of the present invention can be further defined by comparing a cell or cell membrane that has been exposed to a compound to an identical cell or cell membrane preparation that has not been similarly expose to the compound. Alternatively two cells, one containing functional human .beta.1A sodium channel subunit and a second cell identical to the first, but lacking functional human .beta.1A sodium channel subunit could be both used. Both cells or cell membranes are contacted with the same compound and compared for differences between the two cells. This technique is also useful in establishing the background noise of these assays. One of average skill in the art will appreciate that these control mechanisms also allow easy selection of cellular changes that are responsive to modulation of functional human .beta.1A sodium channel subunit.

Particularly preferred cell based assays (or cell membrane assays, if suitable) are those where the cell expresses an endogenous or recombinant sodium .alpha. channel subunit simultaneously with recombinant human .beta.1A. In these assays, a putative modulating compound can be analyzed for its effect on electrophysiological changes to the sodium flux upon the cell for altered expression of .beta.1A expression, or altered expression of the .alpha./.beta.1A complex. Cells expressing recombinant human .beta.1A are subjected to electrophysiological analysis to measure the total influx of sodium ions (Na.sup.30) across the cell membrane by way of voltage, differential using techniques well known by artisans in the field and described herein, including patch clamp voltage techniques as well as membrane proximal voltage sensitive dyes. Compounds that affect the proper function of human .beta.1 may increase or decrease the capacity to open the Na channel, may increase or decrease the rate of Na influx (thus affect the change of membrane potential), may increase or decrease the rate of desensitization or re-sensitization of the channel. The term "test compound" or "modulating compound" as used herein in connection with a suspected modulator of human .beta.1A refers to an organic molecule that has the potential to disrupt specific ion channel activity or cell surface expression of human .beta.1A. For example, but not to limit the scope of the current invention, compounds may include small organic molecules, synthetic or natural amino acid peptides, proteins, or synthetic or natural nucleic acid sequences, or any chemical derivatives of the aforementioned.

The term "cell" refers to at least one cell, but includes a plurality of cells appropriate for the sensitivity of the detection method. Cells suitable for the present invention may be bacterial, yeast, or eukaryotic. For assays to which electrophysiological analysis is conducted, the cells must be eukaryotic, preferably selected from a group consisting of Xenopus oocytes, or PC12, COS-7, CHO, HEK293, SK-N-SH cells.

The assay methods to determine compound modulation of functional human sodium channel .beta.1A subunit can be in conventional laboratory format or adapted for high throughput. The term "high throughput" refers to an assay design that allows easy analysis of multiple samples simultaneously, and capacity for robotic manipulation. Another desired feature of high throughput assays is an assay design that is optimized to reduce reagent usage, or minimize the number of manipulations in order to achieve the analysis desired. Examples of assay formats include 96-well or 384-well plates, levitating droplets, and "lab on a chip" microchannel chips used for liquid handling experiments. It is well known by those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, that greater numbers of samples may be performed using the design of the present invention.

The cellular changes suitable for the method of the present invention comprise directly measuring changes in the function or quantity of human .beta.1A sodium channel subunit, or by measuring downstream effects of human .beta.1A sodium channel subunit function, for example by measuring secondary messenger concentrations or changes in transcription or by changes in protein levels of genes that are transcriptionally influenced by human .beta.1A sodium channel subunit, or by measuring phenotypic changes in the cell. Preferred measurement means include changes in the quantity of human .beta.1A sodium channel subunit protein, changes in the functional activity of human .beta.1A sodium channel subunit, changes in the quantity of mRNA, changes in intracellular protein, changes in cell surface protein, or secreted protein, or changes in Ca+2, cAMP or GTP concentration. Changes in the quantity or functional activity of human .beta.1A sodium channel subunit are described herein. Changes in the levels of mRNA are detected by reverse transcription polymerase chain reaction (RT-PCR) or by differential gene expression. Immunoaffinity, ligand affinity, or enzymatic measurement quantitated changes in levels of protein in host cells. Protein-specific affinity beads or specific antibodies are used to isolate for example .sup.35S-methionine labeled or unlabelled protein. Labeled protein is analyzed by SDS-PAGE. Unlabelled protein is detected by Western blotting, cell surface detection by fluorescent cell sorting, cell image analysis, ELISA or RIA employing specific antibodies. Where the protein is an enzyme, the induction of protein is monitored by cleavage of a fluorogenic or colorimetric substrate.

Preferred detection means for cell surface protein include flow cytometry or statistical cell imaging. In both techniques the protein of interest is localized at the cell surface, labeled with a specific fluorescent probe, and detected via the degree of cellular fluorescence. In flow cytometry, the cells are analyzed in a solution, whereas in cellular imaging techniques, a field of cells is compared for relative fluorescence.

A preferred detection means for secreted proteins that are enzymes such as alkaline phosphatase or proteases, would be fluorescent or colorimetric enzymatic assays. Fluorescent/luminescent/color substrates for alkaline phosphatase are commercially available and such assays are easily adaptable to high throughput multiwell plate screen format. Fluorescent energy transfer based assays are used for protease assays. Fluorophore and quencher molecules are incorporated into the two ends of the peptide substrate of the protease. Upon cleavage of the specific substrate, separation of the fluorophore and quencher allows the fluorescence to be detectable. When the secreted protein could be measured by radioactive methods, scintillation proximity technology could be used. The substrate of the protein of interest is immobilized either by coating or incorporation on a solid support that contains a fluorescent material. A radioactive molecule, brought in close proximity to the solid phase by enzyme reaction, causes the fluorescent material to become excited and emit visible light. Emission of visible light forms the basis of detection of successful ligand/target interaction, and is measured by an appropriate monitoring device. An example of a scintillation proximity assay is disclosed in U.S. Pat. No. 4,568,649, issued Feb. 4, 1986. Materials for these types of assays are commercially available from Dupont NEN.RTM. (Boston, Mass.) under the trade name FlashPlate.TM..

A preferred detection means where the endogenous gene results in phenotypic cellular structural changes is statistical image analysis the cellular morphology or intracellular phenotypic changes. For example, but not by way of limitation, a cell may change morphology such a rounding versus remaining flat against a surface, or may become growth-surface independent and thus resemble transformed cell phenotype well known in the art of tumor cell biology, or a cell may produce new outgrowths. Phenotypic changes that may occur intracellularly include cytoskelet al changes, alteration in the endoplasmic reticulum/Golgi complex in response to new gene transcription, or production of new vesicles.

Where the endogenous gene encodes a soluble intracellular protein, changes in the endogenous gene may be measured by changes of the specific protein contained within the cell lysate. The soluble protein may be measured by the methods described herein.

The present invention is also directed to methods for screening for compounds that modulate the expression of DNA or RNA encoding Human .beta.1A sodium channel subunit as well as the function of Human .beta.1A sodium channel subunit protein in vivo. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding a Human .beta.1A sodium channel subunit, or the function of a Human .beta.1A sodium channel subunit protein. Compounds that modulate the expression of DNA or RNA encoding a Human .beta.1A sodium channel subunit or the function of a Human .beta.1A sodium channel subunit protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Modulators identified in this process are useful as candidate therapeutic agents.

Purification of Human .beta.1A Sodium Channel Subunit Protein

Following expression of the Human .beta.1A sodium channel subunit in a recombinant host cell, the Human .beta.1A sodium channel subunit protein may be recovered to provide the purified Human .beta.1A sodium channel subunit in active form. Several Human .beta.1A sodium channel subunit purification procedures are available and suitable for use. As described above for purification of Human .beta.1A sodium channel subunit from natural sources a recombinant Human .beta.1A sodium channel subunit may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography, lectin chromatography, and antibody/ligand affinity chromatography.

Recombinant Human sodium channel .beta.1A subunits can be separated from other cellular proteins through the use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full length nascent Human .beta.1A sodium channel subunit, polypeptide fragments of Human .beta.1A sodium channel subunit or Human .beta.1A sodium channel subunit subunits. The affinity resin is then equilibrated in a suitable buffer, for example phosphate buffered saline (pH 7.3), and the cell culture supernatants or cell extracts containing a Human .beta.1A sodium channel subunit or Human .beta.1A sodium channel subunit subunits are slowly passed through the column. The column is then washed with the buffer until the optical density (A.sub.280) falls to background, then the protein is eluted by changing the buffer condition, such as by lowering the pH using a buffer such as 0.23 M glycine-HCl (pH 2.6). The purified Human sodium channel .beta.1A subunit protein is then dialyzed against a suitable buffer such as phosphate buffered saline.

Protein Based Assay

The present invention provides an in vitro protein assay method to detect compound modulation of human sodium channel .beta.1A subunit protein activity. The method comprises the steps;

1) contacting a compound and a human .beta.1A sodium channel subunit protein, and

2) measuring a change to human .beta.1A sodium channel subunit function by the compound.

The amount of time necessary for cellular contact with the compound is empirically determined, for example, by running a time course with a known human sodium channel .beta.1A subunit modulator and measuring changes as a function of time.

Production and Use of Antibodies that Bind to Human .beta.1A Sodium Channel Subunit

Monospecific antibodies to Human .beta.1A sodium channel subunit are purified from mammalian antisera containing antibodies reactive against Human .beta.1A sodium channel subunit or are prepared as monoclonal antibodies reactive with Human .beta.1A sodium channel subunit using the technique originally described by Kohler and Milstein, Nature 256: 495 497 (1975). Immunological techniques are well known in the art and described in, for example, Antibodies: A laboratory manual published by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ISBN 0879693142. Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for the human sodium channel .beta.1A subunit. "Homogenous binding" as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the Human .beta.1A sodium channel subunit, as described above. Human .beta.1A sodium channel subunit specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of Human .beta.1A sodium channel subunit either with or without an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.001 mg and about 1000 mg of human sodium channel .beta.1A subunit associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The initial immunization consists of human sodium channel .beta.1A subunit in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at about three-week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20.degree. C.

Monoclonal antibodies (mAb) reactive with Human .beta.1A sodium channel subunit are prepared by immunizing inbred mice, preferably Balb/c, with Human .beta.1A sodium channel subunit. The mice are immunized by the IP or SC route with about 0.001 mg to about 1.0 mg, preferably about 0.1 mg, of Human .beta.1A sodium channel subunit in about 0.1 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's adjuvant is preferred, with Freund's complete adjuvant being used for the initial immunization and Freund's incomplete adjuvant used thereafter. The mice receive an initial immunization on day 0 and are rested for about 2 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.001 to about 1.0 mg of Human .beta.1A sodium channel subunit in a buffer solution such as phosphate buffered saline by the intravenous (IV) route.

Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions that will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp2/0, with Sp2/0 being generally preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA). or ELISA using Human .beta.1A sodium channel subunit as the antigen. The culture fluids can also be tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973 or by the technique of limited dilution.

Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 1.times.10.sup.6 to about 6.times.10.sup.6 hybridoma cells at least about 4 days after priming. Ascites fluid is collected at approximately 8 12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-Human .beta.1A sodium channel subunit mAb is carried out by growing the hybridoma in tissue culture media well known in the art. High density in vitro cell culture may be conducted to produce large quantities of anti-human .beta.1A sodium channel subunit mAbs using hollow fiber culture techniques, air lift reactors, roller bottle, or spinner flasks culture techniques well known in the art. The mAb are purified by techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of Human .beta.1A sodium channel subunit in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for Human .beta.1A sodium channel subunit polypeptide fragments, or full-length nascent Human .beta.1A sodium channel subunit polypeptide, or the individual Human .beta.1A sodium channel subunit subunits. Specifically, it is readily apparent to those skilled in the art that monospecific antibodies may be generated which are specific for only one Human .beta.1A sodium channel subunit or the fully functional human .beta.1A sodium channel subunit protein. It is also apparent to those skilled in the art that monospecific antibodies may be generated that inhibit normal function of human .beta.1A sodium channel subunit protein.

Human .beta.1A sodium channel subunit antibody affinity columns are made by adding the antibodies to a gel support such that the antibodies form covalent linkages with the gel bead support. Preferred covalent linkages are made through amine, aldehyde, or sulfhydryl residues contained on the antibody. Methods to generate aldehydes or free sulfhydryl groups on antibodies are well known in the art; amine groups are reactive with, for example, N-hydroxysuccinimide esters.

Kit Compositions Containing Human .beta.1A Sodium Channel Subunit Specific Reagents

Kits containing Human .beta.1A sodium channel subunit DNA or RNA, antibodies to Human .beta.1A sodium channel subunit, or Human .beta.1A sodium channel subunit protein may be prepared. Such kits are used to detect DNA which hybridizes to Human .beta.1A sodium channel subunit DNA or to detect the presence of Human .beta.1A sodium channel subunit protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including, but not limited to, forensic analyses, diagnostic applications, and epidemiological studies.

The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of Human .beta.1A sodium channel subunit DNA, Human .beta.1A sodium channel subunit RNA or Human .beta.1A sodium channel subunit protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of Human .beta.1A sodium channel subunit. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant Human .beta.1A sodium channel subunit protein or anti-human .beta.1A sodium channel subunit antibodies suitable for detecting Human .beta.1A sodium channel subunit. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.

Gene Therapy

Nucleotide sequences that are complementary to the Human .beta.1A sodium channel subunit encoding DNA sequence can be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O-alkylRNA, or other Human .beta.1A sodium channel subunit antisense oligonucleotide mimetics. Human .beta.1A sodium channel subunit antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. Human .beta.1A sodium channel subunit antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce Human .beta.1A sodium channel subunit activity.

Human .beta.1A sodium channel subunit gene therapy may be used to introduce Human .beta.1A sodium channel subunit into the cells of target organisms. The Human .beta.1A sodium channel subunit gene can be ligated into viral vectors that mediate transfer of the Human .beta.1A sodium channel subunit DNA by infection of recipient host cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like. Alternatively, Human .beta.1A sodium channel subunit DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo Human .beta.1A sodium channel subunit gene therapy. Human .beta.1A sodium channel subunit gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate Human .beta.1A sodium channel subunit activity. Protocols for molecular methodology of gene therapy suitable for use with the human .beta.1A sodium channel subunit gene is described in Gene Therapy Protocols, edited by Paul D. Robbins, Human press, Totawa N.J., 1996.

Pharmaceutical Compositions

Pharmaceutically useful compositions comprising Human .beta.1A sodium channel subunit DNA, Human .beta.1A sodium channel subunit RNA, or Human .beta.1A sodium channel subunit protein, or modulators of Human .beta.1A sodium channel subunit receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modulator.

Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders in which modulation of Human .beta.1A sodium channel subunit-related activity is indicated. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.

The term "chemical derivative" describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the Human .beta.1A sodium channel subunit receptor or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds or modulators identified according to this invention as the active ingredient for use in the modulation of Human .beta.1A sodium channel subunit can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as a Human .beta.1A sodium channel subunit modulating agent.

The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. The dosages of the Human .beta.1A sodium channel subunit receptor modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

Advantageously, compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or .beta.-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents that may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations, which generally contain suitable preservatives, are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds or modulators of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

For oral administration, the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed. The capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained. An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection. The active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed. Alternatively the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal. Suitable inert carriers include corn meal, citrus meal, fermentation residues, soya grits, dried grains and the like. The active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling, or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.

The compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraluminal, intratracheal, or subcutaneous. The injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like. As an alternative, aqueous parenteral formulations may also be used. The vegetable oils are the preferred liquid carriers. The formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension. These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent. Formulations containing from 0.005 to 10% by weight of the active ingredient are acceptable. Preferred formulations are those containing from 0.01 to 5% by weight of the instant compounds or modulators.


Claim 1 of 3 Claims

1. A method of screening for a modulator of sodium channel activity comprising: (a) providing a cell that co-expresses a protein encoded by SEQ ID NO:12 and a sodium channel .alpha. subunit protein, wherein the cell elicits a sodium ion flux; (b) contacting the cell with a putative .beta.1A modulating compound; (c) measuring a change upon the cell that alters the sodium ion flux; and (d) comparing said change to a base value observed in an otherwise identical cell that does not express said encoded protein.

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