Screening assays for identifying agents that modulate BK channel activity and further modulate the sleep/wake cycle in a subject, circadian regulated locomotor activity in a subject, or both are provided, as are agents identified using such screening assays. Also provided are methods of modulating the sleep/wake cycle in a subject and methods of modulating circadian regulated locomotor activity in a subject by administering an agent that modulates BK channel activity to the subject, for example, an agent identified by a screening assay as disclosed.

Description of the Invention

Most organisms have endogenous biological clocks that coordinate physiology and behavior to adapt to diurnal changes in the environment. In mammals, the suprachiasmatic nucleus (SCN) is the anatomical site of a master pacemaker that regulates rhythmic processes throughout the body. Recent work indicates that peripheral circadian clocks also exist, suggesting they may exert proximal regulation of physiology specific to their target tissues. In Drosophila, a number of key processes such as emergence from the pupal case, locomotor activity, feeding, and aspects of mating behavior are under circadian regulation. Although the mechanisms by which the molecular oscillations take place are generally understood, a clear link between gene regulation and downstream biological processes has not previously been established.

An oligonucleotide-based high density array that interrogates gene expression changes on a whole genome level was used to identify clock controlled output genes in Drosophila and in mice. As disclosed herein, a variety of physiological processes ranging from protein stability and degradation, signal transduction, heme metabolism and detoxification were found to be under circadian transcriptional regulation in Drosophila (Example 1; see, also, Panda et al., Nature 417:329-335, 2002a, which is incorporated herein by reference). A comparison of rhythmically expressed genes in the fly head and body revealed that the clock has adapted its output functions to the needs of each particular tissue, thus indicating that tissue-specific gene expression is superimposed on clock control of gene expression. A cycling calcium dependent potassium channel protein, slowpoke (slo) was identified as providing a key step in linking the transcriptional feedback loop to rhythmic locomotor behavior. As disclosed herein, expression of slo correlated with regulation of the sleep/wake cycle in Drosophila.

Examination of circadian output genes in the mouse SCN, which includes the central clock, and in mouse liver, which is an important regulator of physiology, revealed that approximately 10% of detectably expressed transcripts in both tissues were under circadian control, and that almost all of these output genes were specific to either the SCN or liver (Example 2, see Original Patent). In addition, twenty proteins were identified that cycled in both the head and body (Table 5, see Original Patent). The cycling of these proteins in the head and body indicates they represent basic clock components. Genes encoding proteins in rate-limiting steps in pathways involved in endocrine regulation of physiology, energy metabolism, and the redox state of the cell, and genes coding for both intracellular and extracellular signaling components were clock regulated. Remarkably, clock-regulated expression of the Kcnma1 gene, which encodes a calcium activated potassium channel orthologous to that encoded by the Drosophila slo gene, also was identified in mouse SCN. These results indicate that cyclic potassium channel activity is involved in the coordination of the rhythmic locomotor activity associated with the sleep/wake cycle, in eukaryotic organisms.

Clusters of genes involved in specific various biological pathways were identified as being coordinately expressed in a circadian-regulated manner in Drosophila and in the mouse (see Examples 1 and 2). Furthermore, when circadian-regulated expression of gene clusters was examined in the fly head as compared to fly bodies, only a few transcripts cycled in both tissues (Example 1; see, also, Ceriani et al., J. Neurosci. 22:9305-9319, 2002, which is incorporated herein by reference). Similar results were obtained when cycling transcripts in the mouse SCN were compared to those cycling in mouse liver (Example 2; see, also, Table 5 (see Original Patent); see, also, Panda et al., Cell 109:307-320, 2002b, which is incorporated herein by reference). However, while common cycling transcripts in central (head) as compared to peripheral tissues were rare, many transcripts that cycled, for example, in fly heads also were expressed in the fly bodies, indicating that differential transcriptional regulation occurs with respect to these genes.

One of the fly genes identified to be circadian-regulated in fly heads, but not in fly bodies, was that encoding the slowpoke binding protein, slob, which binds to the calcium ion-dependent voltage gated potassium channel, slowpoke (see Example 1). McDonald and Rosbash (supra, 2001) reported circadian cycling of slob expression, and suggested that cycling slob, through its interaction with slo, could give rise to circadian oscillations in potassium channel activity. The authors further suggested that such circadian oscillations in potassium channel activity could affect resting membrane potential, which, in turn, would result in calcium ion oscillations that may underlie oscillations in neuropeptide staining reported in lateral neuron termini (Id.). Circadian cycling of slob expression also was reported by Claridge-Chang et al. (supra, 2001), who suggested that oscillating slob protein, through its interaction with slo, may be involved in rhythmic control of synaptic function, including synaptic plasticity, a process that may require sleep. Claridge-Chang et al. further demonstrated by in situ hybridization that slob mRNA expression occurred in the developing mushroom body of the fly larval brain, and corresponded with the region of larval brain receiving projections of lateral neurons (LNs), which comprise the circadian pacemaker cells, and, based on these observations, suggested that innervating LNs may be required for cycling slob expression (Id.).

Neither McDonald and Rosbash (supra, 2001) nor Claridge-Chang et al. (supra, 2001) reported whether expression of slo mRNA correlated with slo protein expression or whether slo protein levels cycle in fly brain. It is well recognized that mRNA expression does not necessarily correlate with translation of an encoded protein in cells, and even when mRNA is translated, there is not necessarily a correlation between the level of mRNA in the cells and the amount of protein generated. For example, the mammalian protein HIF-1.alpha. is constitutively expressed at the mRNA level, however, HIF-1.alpha. protein only is apparent following exposure to low oxygen conditions (Proc. Natl. Acad. Sci., USA 92:5510-5514, 1995). Furthermore, neither of the references correlate slo mRNA expression with rhythmic locomotor activity in anticipation of dusk and dawn or with regulation of the sleep/wake cycle.

As disclosed herein, slob mRNA cycled robustly in the heads of flies exposed to either entrained (LD) or free running (DD) conditions, peaking at about ZT18, but did not cycle in clk.sup.jrk flies, which are mutants that have impaired clock function (Example 1, see Original Patent). Based on this result, expression of slo was examined and found to oscillate in phase with slob, with a peak expression at ZT20 (see Ceriani et al., supra, 2002; FIGS. 4A to 4C, see Original Patent). Immunocytochemical analysis of whole fly brain mounts revealed that the slo protein was localized in a subset of the ventral LNs. In wild type flies, cycling of slo correlated with locomotor activity, which increased in anticipation of dawn and dusk. In comparison, flies containing slo mutations failed to show a change in activity in anticipation of dusk and dawn, even though total activity for wild type and slo mutant flies was approximately the same (see Ceriani et al., supra, 2002; FIG. 5, see Original Patent). As such, the present disclosure extends previous observations by demonstrating that slo, regulates changes in locomotor activity in anticipation of dawn and dusk, thus indicating that slo is a key regulator of the sleep/wake cycle. Furthermore, clock regulated expression of the Kcnma1 gene, which is a slo ortholog in mice, paralleled that found in Drosophila (see Example 2), indicating that the factors regulating the sleep/wake cycle are evolutionarily conserved. Accordingly, the present invention provides methods of modulating the sleep/wake cycle and methods of modulating circadian regulated locomotor activity in an individual by increasing or decreasing calcium activated potassium channel ("BK channel") levels or activity; drug screening assays, which allow the identification of agents that increase or decrease BK channel activity and, therefore, can modulate circadian regulated locomotor activity or the sleep wake cycle; and agents identified using such a screening assay.

In addition to clock regulated BK channel gene expression, which is localized to the head, several genes that cycle in a circadian regulated manner in both the suprachiasmatic nucleus and liver of mice were identified (Table 5). The sequences of the polypeptides can be obtained by searching for the appropriate identifier in the NCBI database ("Refseq" in Table 5) or in the Unigene database ("Unigene" in Table 5), which also provides ready access to orthologs of the listed mouse polypeptides. The more generalized rhythmic cycling of the clock regulated proteins listed in Table 5 indicates that they represent basic components of the circadian clock. As such, these rhythmically cycling proteins, as well as the genes encoding them (particularly the regulatory elements of such genes), provide additional targets useful in screening assays to identify agents that can modulate the circadian clock in an organism.

Screening assays of the invention are exemplified herein with reference to the BK channel. It will be recognized, however, that screening assays of the invention also can be performed using one or more of the proteins shown in Table 5, except that the methods will utilize an assay useful for detecting a change in the activity or function of the particular protein or proteins used in the assay. For example, where the protein used in a screening assay of the invention is a kinase such as casein kinase 1, alpha 1 or adenylate kinase or a phosphatase such as protein tyrosine phosphatase, non-receptor type 16 or protein tyrosine phosphatase 4a1 (see Table 5), the screening assay will comprise contacting the kinase or phosphatase with a substrate for the kinase or phosphatase, and an agent to examined for the ability to modulate the kinase or phosphatase activity, wherein a change in such activity due to the agent identifies the agent as one that can modulate a circadian function in a subject expressing the protein in a clock regulated manner. In one embodiment, a screening assay of the invention comprises a high throughput assay, wherein at least two of the polypeptides of Table 5, including, if desired, the mouse BK channel and/or the mouse Per2 gene product, or at least two regulatory elements comprising the 5' untranslated regions of the gene sequences encoding such polypeptides, can be contacted in parallel with one or more agents to be examined for an ability to modulate the activity of one or more of the polypeptides (or regulatory elements), thus providing a means to identify an agent that modulates the activity or level of expression of one or more clock regulated proteins. If desired, such an agent then can be examined, for example, for an ability to modulate the sleep/wake cycle or other clock regulated biochemical or physiological activity of a subject.

Accordingly, in one embodiment, the a screening assay of the invention provides means of identifying agents that can modulate the sleep/wake cycle in a subject. As exemplified herein, a screening assay of the invention can be performed, for example, by contacting a test system, which includes a BK channel, with an agent suspected of having the ability to modulate the sleep/wake cycle in the subject; detecting a change in activity of the BK channel in the presence of the agent as compared to the activity of the BK channel in the absence of the agent, thereby identifying an agent that modulates BK channel activity; administering the agent that modulates BK channel activity to a test subject; and detecting a change in the sleep/wake cycle of the test subject due to administration of the agent that modulates BK channel activity due to administration of the agent.

In another embodiment, a screening assay of the invention provides a means of identifying agents that can modulate circadian regulated locomotor activity in a subject. Such a method can be performed, for example, by contacting a test system containing a BK channel with an agent suspected of having the ability to modulate circadian regulated locomotor activity in the subject; detecting a change in activity of the BK channel in the presence of the agent as compared to the activity of the BK channel in the absence of the agent, thereby identifying an agent that modulates BK channel activity; administering the agent that modulates BK channel activity to a test subject; and detecting a change in circadian regulated locomotor activity of the test subject due to administration of the agent.

As used herein, the term "BK channel" or "calcium activated potassium channel" refers to the high conductance channel that is present in neuronal tissue and smooth muscle of eukaryotic organisms and is gated by intracellular calcium ion concentration and membrane potential. For purposes of the present invention, a BK channel comprises at least a Drosophila slo polypeptide, or a homolog, ortholog, or paralog thereof (collectively "wild type" slo or BK channel), or a variant of a wild type slo polypeptide (e.g., mutant). Such channels, which, in organisms such as mammals, can contain an .alpha. subunit and a .beta. subunit, also are referred to as "maxi-K channels", enable efflux of potassium ions when opened due to an increase in the intracellular calcium ion concentration or membrane depolarization (change in potential). A BK channel useful in a drug screening assay of the invention can be a BK channel of any species, preferably a eukaryotic species, including an invertebrate such as an insect or a nematode, or a vertebrate such as an amphibian, avian or mammalian species.

BK channels are well known in the art and exemplified by those encoded by the Drosophila slowpoke (slo) gene, as well as by eukaryotic orthologs of slo, including mammalian slo (also referred to as Kcnma1) gene products, for example, the mouse slo (mslo) and human slo (hslo) orthologs. The nucleotide and amino acid sequences of Drosophila slo (Atkinson et al., Science 253:551, 1991; Adelman et al., Neuron 9:209, 1992; GenBank Acc. No. NM.sub.--079762, each of which is incorporated herein by reference) are well known, as are those of orthologs such as the mouse Kcnma1 ortholog (Butler et al., Science 261:221, 1993; Pallanck and Ganetzsky, Hum. Mol. Genet. 3:1239, 1994; GenBank Acc. No. NM.sub.--010610, each of which is incorporated herein by reference), human Kcnma1 (see Butler et al., supra, 1993; Pallanck and Ganetzsky, supra, 1994; see, also, Dworetzky et al., Brain Res. Mol. Brain. Res. 27:189, 1994; Tsang-Crank et al., Neuron 13:1315, 1994; GenBank Acc. No. NM.sub.--002247, each of which is incorporated herein by reference), rat Kcnma1 (see, for example, GenBank Acc. No. NM.sub.--031828, which is incorporated herein by reference); rabbit Kcnma1 (see, for example, GenBank Ace. No. AF321818, which is incorporated herein by reference). In view of the conserved sequence homology of the exemplified Drosophila and mammalian BK channel nucleotide and amino acid sequences, it will be recognized that other wild type slo polynucleotides and variants thereof readily can be identified and used in the methods of the invention.

The BK channel (or other clock regulated gene product) used in a screening assay of the invention can be in an isolated form, for example, a BK channel expressed from a recombinant nucleic acid or generated using an in vitro translation or coupled transcription/translation reaction. An isolated BK channel also can be obtained from cells that normally express the channel using routine methods for isolating a polypeptide from a membrane fraction of cells or can be generated using chemical synthesis methods. As used herein, the term "substantially purified" or "isolated", when used in reference to a polypeptide or a polynucleotide, means that the polypeptide or polynucleotide is in a form other than that in which it exists in nature. In general, an isolated polypeptide or polynucleotide is relatively free of materials with which it is naturally associated with in a cell. For example, a substantially purified clock regulated gene product such as a BK channel can comprise at least about 10% of a mixture, generally at least about 25% of a mixture, usually at least about 50% of a mixture, and particularly about 90% or more of a mixture containing the polypeptide. A determination that a polypeptide or a polynucleotide is substantially purified can be made using well known methods, for example, by performing electrophoresis and identifying the particular molecule as a relatively discrete band. A substantially purified polynucleotide, for example, can be obtained by cloning the polynucleotide, or by chemical or enzymatic synthesis. A substantially purified polypeptide can be obtained, for example, by a method of chemical synthesis, or using methods of protein purification, followed by proteolysis and, if desired, further purification by chromatographic or electrophoretic methods.

It should be recognized, however, that an isolated BK channel polypeptide, for example, can be added to a reaction mixture or that an isolated polynucleotide encoding a BK channel polypeptide can be introduced into a cell. Nevertheless, the polypeptide or polynucleotide is considered to be (or have been) substantially purified because it is not in the form in which it exists in nature. Methods for isolating a polypeptide are well known and include, for example, extraction, precipitation, ion exchange chromatography, affinity chromatography, and gel filtration methods, including combinations of such methods. For example, a BK channel can isolated by affinity chromatography using an antibody or other protein that specifically binds the BK channel. BK channel binding proteins are disclosed herein and well known in the art.

A test system for practicing a method of the invention can contain a substantially purified BK channel polypeptide, and contacting with the agent can be performed in vitro, for example, in a test tube, in a well of a plate, or in a defined position on a microchip, thus allowing for high throughput screening of agents suspected of having the ability to modulate BK channel activity. Such an in vitro reaction generally is performed in an aqueous solution, which can contain buffers that maintain the reaction at a desired pH; salts such as those providing potassium ions and calcium ions; and other reagents useful for performing such a reaction.

A BK channel used in a method of the invention also can be contained in a membrane, including a synthetic membrane or an isolated naturally occurring membrane; or a membrane of an intact cell that normally expresses the BK channel or that has been genetically modified to express the BK channel. The membrane can be a synthetic membrane, for example, a liposome or a synthetic lipid bilayer. Generally, though not necessarily, a BK channel will traverse the cell membrane, in which case the cell membrane has a first side and a second side. Depending on the assay being performed, the first and second side can be opposite side of a surface formed by the membrane, or can be an interior side and an exterior side of a membrane that forms an enclosed volume.

A membrane containing a BK channel and useful in a method of the invention also can be a cell membrane that has been isolated from a cell. The cell membrane can be obtained from a cell that naturally expresses the BK channel, for example, a cell membrane isolated from a muscle cell or a nerve cell of a eukaryotic organism such as a mammal. The cell membrane also can be isolated from a cell that has been genetically modified to express a heterologous BK channel. In a cell membrane obtained from such a genetically modified cell, the heterologous BK channel can be the only BK channel expressed in the cell membrane, or can be co-expressed with an endogenous BK channel. Furthermore, in this and other aspects of a screening method of the invention, the BK channel can be a wild type BK channel or a mutant BK channel (see, for example, Elkins et al., Proc. Natl. Acad. Sci., USA 83:8415, 1986, which is incorporated herein by reference).

A drug screening assay of the invention also can be practiced using an intact cell, which is delimited by a cell membrane containing a BK channel. The cell can be a muscle cell, nerve cell, kidney cell, epithelial cell, or other cell that expresses an endogenous BK channel, or can be cell that is genetically modified to express a heterologous BK channel from a polynucleotide introduced into the cell or into a cell from which used in the assay is derived. As used herein, the term "genetically modified" refers to a cell containing a heterologous polynucleotide that has been introduced into the cell using a recombinant DNA method. The term "heterologous" is used herein to indicate that a polynucleotide or polypeptide is not endogenous to a cell (or isolated cell membrane) in which it is introduced or contained, or that the polynucleotide or polypeptide is part of a construct such that it is in a form other than it normally would be found in a cell. As such, a polynucleotide, for example, encoding a BK channel that is introduced into a cell is heterologous with respect to the cell, as is a polypeptide expressed therefrom. It should be recognized that such a heterologous polynucleotide or polypeptide can be identical to an endogenous polynucleotide or polypeptide that also can naturally be present in the cell; for example, an expressible mouse slo polynucleotide can be introduced into mouse muscle cells that express endogenous mouse slo polypeptide, such that the number of slo polypeptides expressed in the cell is increased, thus providing a means to increase the sensitivity of a screening assay of the invention.

A heterologous polynucleotide, which can encode a wild type or mutant BK channel polypeptide or BK channel binding protein, a reporter polypeptide, or other polypeptide as desired, can be transiently expressed in the genetically modified cell, or can be stably maintained in the cell. The polynucleotide can be contained in a vector, which can facilitate manipulation of the polynucleotide, including introduction of the polynucleotide into a target cell. The vector can be a cloning vector, which is useful for maintaining the polynucleotide, or can be an expression vector, which contains, in addition to the polynucleotide, regulatory elements useful for transcription and, where appropriate, translation of the polynucleotide. An expression vector can contain the expression elements necessary to achieve, for example, sustained transcription of the encoding polynucleotide, or the regulatory elements can be operatively linked to the polynucleotide prior to its being cloned into the vector. For example, the polynucleotide can be operatively linked to a tissue specific regulatory element, for example, a muscle cell specific regulatory element, such that expression of an encoded polypeptide is restricted to muscle cells. Muscle cell specific regulatory elements including, for example, the muscle creatine kinase promoter (Sternberg et al., Mol. Cell. Biol. 8:2896, 1988, which is incorporated herein by reference) and the myosin light chain enhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA 88:5847, 1991, which is incorporated herein by reference) are well known in the art.

An expression vector (or the polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive, inducible, tissue specific, or developmental stage specific expression of the encoding polynucleotide, a poly-A recognition sequence, and a ribosome recognition site or internal ribosome entry site, or other regulatory elements such as an enhancer, which can be tissue specific. The vector also can contain elements required for replication in a prokaryotic or eukaryotic host system or both, as desired. Such vectors, which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51, 1994; Flotte, J. Bioenerg. Biomemb. 25:37, 1993; Kirshenbaum et al., J. Clin. Invest. 92:381, 1993; each of which is incorporated herein by reference).

Viral expression vectors can be particularly useful for introducing a polynucleotide into a cell, including, where desired, a cell in a subject. Viral vectors provide the advantage that they can infect host cells with relatively high efficiency and can infect specific cell types. For example, a polynucleotide encoding a BK channel polypeptide such as Drosophila slo, mouse slo, human slo, or the like, can be cloned into a baculovirus vector, which then can be used to infect an insect host cell, thereby providing a means to produce large amounts of the encoded slo polypeptide. The viral vector also can be derived from a virus that infects cells of an organism of interest, for example, vertebrate host cells such as mammalian, avian or piscine host cells. Viral vectors have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like (see Miller and Rosman, BioTechniques 7:980, 1992; Anderson et al., Nature 392:25, Suppl., 1998; Verma and Somia, Nature 389:239, 1997; Wilson, New Engl. J. Med. 334:1185 (1996), each of which is incorporated herein by reference).

A polynucleotide encoding a clock regulated gene product such as a BK channel, or encoding a polypeptide that specifically interacts with the clock regulated gene product and is required for or facilitates its activity, for example, a BK channel binding protein, or encoding a reporter molecule, selectable marker, or the like, which can, but need not, be contained in a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1987, and supplements through 1995), each of which is incorporated herein by reference). Such methods include, for example, transfection, lipofection, microinjection, electroporation and, with viral vectors, infection; and can include the use of liposomes, microemulsions or the like, which can facilitate introduction of the polynucleotide into the cell and can protect the polynucleotide from degradation prior to its introduction into the cell. Accordingly, a polynucleotide can be introduced into a cell as a naked nucleic acid molecule, can be incorporated in a matrix such as a liposome or a particle such as a viral particle, or can be incorporated into a vector. The selection of a particular method will depend, for example, on the cell into which the polynucleotide is to be introduced, as well as whether the cell is isolated in culture, or is in a tissue or organ in culture or in situ.

Introduction of a polynucleotide into a cell by infection with a viral vector is particularly advantageous in that it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo (see, for example, U.S. Pat. No. 5,399,346, which is incorporated herein by reference). Moreover, viruses are very specialized and can be selected as vectors based on an ability to infect and propagate in one or a few specific cell types. Thus, their natural specificity can be used to target the polynucleotide contained in the vector to specific cell types. As such, a vector based on an HIV can be used to infect T cells, a vector based on an adenovirus can be used, for example, to infect respiratory epithelial cells, a vector based on a herpesvirus can be used to infect neuronal cells, and the like. Other vectors, such as adeno-associated viruses can have greater host cell range and, therefore, can be used to infect various cell types, although viral or non-viral vectors also can be modified with specific receptors or ligands to alter target specificity through receptor mediated events.

Generally, a polynucleotide encoding a clock regulated gene product, for example, a BK channel, is introduced into a cell with a polynucleotide encoding a selectable marker, which provides a means to select cells that contain the introduced polynucleotide. Selectable markers include, for example, those that confer antimetabolite resistance, for example, dihydrofolate reductase, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13:143-149, 1994); neomycin phosphotransferase, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO. J. 2:987-995, 1983) and hygro, which confers resistance to hygromycin (Marsh, Gene 32:481-485, 1984), trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627); ornithine decarboxylase, which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine (DFMO; McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.); and deaminase from Aspergillus terreus, which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59:2336-2338, 1995). In addition, reporter molecules can act as markers that facilitate identification of a plant cell containing the polynucleotide encoding the marker include, for example, luciferase (Giacomin, Plant Sci. 116:59-72, 1996; Scikantha, J. Bacteriol. 178:121, 1996), green fluorescent protein (Gerdes, FEBS Lett. 389:44-47, 1996), and numerous others as disclosed herein or otherwise known in the art.

A cell expressing a BK channel can be contacted with a test agent ex vivo, for example, in a cell culture or in a tissue or organ culture. As disclosed herein, the cell can be a eukaryotic cell, for example, a mammalian cell such as a human nerve cell or muscle cell, which naturally expresses a BK channel, or a cell that is genetically modified to express a BK channel, for example, a Xenopus oocyte, which can be genetically modified to express a wild type or variant slo polypeptide (see, for example, U.S. Pat. No. 5,637,470). The cell to be contacted also can be present in situ in an organism, which can, but need not, be a transgenic organism containing cells expressing, for example, a heterologous BK channel.

In a screening assay of the invention, the test system, which can be a reaction mixture containing an isolated BK channel polypeptide or an isolated cell membrane, or an intact cell, can further contain a BK channel binding protein, for example, a Drosophila slo binding protein (slob), which is encoded by slob (GenBank Ace. No. AY060721; Schopperle et al., Neuron 20:565, 1998, each of which is incorporated herein by reference), or a homolog, ortholog or paralog of Drosophila slob, or a variant thereof. In some organisms such as mammals, the BK channel is formed as a heterodimer, including an .alpha. subunit, which is an ortholog of Drosophila slo and comprises the pore forming unit, and a .beta. subunit, which has a regulatory activity and, for purposes of the present invention, is considered a BK channel binding protein. Thus, where a screening assay of the invention utilizes a mammalian slo protein, for example, a human slo protein, the assay can further include the BK channel .beta. subunit, i.e., a human .beta. subunit. Nucleotide and amino acid sequences encoding mammalian BK channel .beta. subunits are well known (see, for example, U.S. Pat. No. 5,637,470; Meera et al., FEBS Lett. 382:84, 1996, each of which is incorporated herein by reference).

Although mammalian BK channels can comprise a heterodimer, it should be recognized that inclusion of the BK channel binding protein (i.e., .beta. subunit) is not necessary for practicing a screening assay of the invention. For example, when mouse slo was expressed alone in Xenopus oocytes, large conductance, potassium ion selective channel activity characteristic of BK channels was observed, and the activity was sensitive to charybdotoxin (CbTX) and iberiotoxin (ITX), which are selective for BK channels (Butler et al., supra, 1993). In other experiments, oocytes genetically modified to express a human .beta. subunit, alone, exhibited no measurable potassium currents different from those in control oocytes using whole oocyte and patch-clamp recording methods, whereas oocytes expressing only the human .alpha. subunit (hslo) exhibited large outward currents that were activated at positive membrane potentials, and blocked by CbTX and ITX (U.S. Pat. No. 5,637,470). Oocytes genetically modified to express the human .alpha. and human .beta. subunits also exhibited outward potassium currents that were blocked by CbTX and ITX. The magnitudes of currents were similar to those observed in oocytes expressing only the .alpha. subunit. However, the outward currents in oocytes expressing the .alpha. and .beta. subunits were activated at more negative potentials than oocytes expressing only the .alpha. subunit, and were activated by a BK channel activator that did not activate the channel in oocytes expressing only the .alpha. subunit (U.S. Pat. No. 5,637,470; see, also, Meera et al., supra, 1996). Thus, in a screening assay for identifying an agent that modulates the activity of a BK channel that comprises an .alpha. subunit and .beta. subunit in nature, there can be advantages to practicing a method of the invention with a test system containing the BK channel (.alpha. subunit) and BK channel binding protein (.beta. subunit). It will be recognized, however, that not all BK channel binding proteins bind all BK channels. For example, Drosophila slob binds Drosophila slo but does not bind mouse slo (Schopperle et al., supra, 1998), whereas human slo binds the human .beta. subunit and the bovine .beta. subunit, both of which up-regulate the slo (.alpha. subunit) channel activity (Meera et al., supra, 1996).

In view of the exemplified polynucleotides and encoded BK channel and BK channel binding protein polypeptides, it will be recognized that well known procedures and algorithms based on identity (or homology) to the exemplified sequences can be used to identify homologs, orthologs, and variants thereof useful in the screening methods of the invention (see, for example, U.S. Pat. No. 5,966,712, which is incorporated herein by reference). Such polynucleotides, for example, can be identified by performing a BLASTN search using the Drosophila slo or murine kcnma1 polynucleotides as query sequences and selecting those substantially similar sequences, for example, sequences having an E value.ltoreq.1.times.10.sup.-8. Such homologous, orthologous or variant polynucleotides can be useful for performing the screening assays of the invention.

As used herein, the term "substantially similar", when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference polynucleotide, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference polynucleotide. For purposes of the present invention, a reference (or query) sequence is a polynucleotide encoding a BK channel or a BK channel binding protein. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between the substantially similar nucleotide sequence and the reference polynucleotide is at least 60%, generally at least 75%, particularly at least 90%, preferably at least 95%, and more preferably at least 99%. A nucleotide sequence "substantially similar" to a reference polynucleotide can selectively hybridize to the reference polynucleotide, but not to an unrelated polynucleotide, under hybridization conditions such as provided by incubation in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree. C.; generally by incubation in 7% SDS, 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC, 0.1% SDS at 50.degree. C.; particularly by incubation in 7% SDS, 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.; preferably by incubation in 7% SDS, 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C.; and more preferably by incubation in 7% SDS, 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

The term "substantially similar," when used in reference to a polypeptide sequence, means that an amino acid sequence relative to a reference (query) sequence shares at least about 65% amino acid sequence identity, generally at least about 75% amino acid sequence identity, particularly at least about 85%, preferably at least about 90%, and more preferably at least about 95% or greater amino acid sequence identity. Generally, sequences having an E.ltoreq.10.sup.-8 are considered to be substantially similar to a query sequence. Such sequence identity can take into account conservative amino acid changes that do not substantially affect the function of a polypeptide. As such, homologs or orthologs of the Drosophila and murine circadian-regulated genes, particularly Drosophila slo and murine kcnma1, variants thereof, and polypeptides (and encoding polynucleotides) substantially similar to those exemplified herein can be used for practicing the methods of the invention.

Homology or identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. The terms "homology" and "identity," when used herein in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues, respectively, that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.

For sequence comparison, one sequence generally acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

The term "comparison window" is used broadly herein to include reference to a segment of any one of the number of contiguous positions, for example, about 20 to 600 positions, for example, amino acid or nucleotide position, usually about 50 to about 200 positions, particularly about 100 to about 150 positions, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequence for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Person and Lipman (Proc. Natl. Acad. Sci., USA 85:2444, 1988), each of which is incorporated herein by reference; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by manual alignment and visual inspection. Other algorithms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local Content Program), MACAW (Multiple Alignment Construction & Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment by Genetic Algorithm) and WHAT-IF (see, also, U.S. Pat. No. 5,966,712). Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences to a polynucleotide encoding a BK channel polypeptide or BK channel binding protein.

A number of genome databases are available for comparison. Several databases containing genomic information annotated with some functional information are maintained by different organizations, and are accessible on the world wide web via the internet, for example, at the URLs "wwwtigr.org/tdb"; "genetics.wisc.edu"; "genome-www.stanford.edu/.about.ball"; "hiv-web.lanl.gov"; "ncbi.nlm.nih.gov"; "ebi.ac.uk"; "Pasteur.fr/other/biology"; and "genome.wi.mit.edu". In particular, sequences and expression characteristics of the circadian regulated expression of Drosophila and mouse genes as disclosed herein are accessible on the world wide web at the URL "expression.gnf.org/circadian".

The BLAST and BLAST 2.0 algorithms using default parameters are particularly useful for identifying polynucleotides encoding polypeptides substantially similar to the exemplified BK channel polypeptides and BK channel binding proteins (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977; J. Mol. Biol. 215:403-410, 1990, each of which is incorporated herein by reference). Software for performing BLAST analyses is publicly available on the world wide web through the National Center for Biotechnology Information at the URL "ncbi.nlm.nih.gov". This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra, 1977, 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci., USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci., USA 90:5873, 1993, which is incorporated herein by reference). One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

Protein and nucleic acid sequence homologies can be evaluated using the Basic Local Alignment Search Tool ("BLAST"). In particular, five specific BLAST programs can be used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993, each of which is incorporated herein by reference). Less preferably, the PAM or PAM250 matrices may also be used (Schwartz and Dayhoff, eds., "Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure" (Washington, National Biomedical Research Foundation 1978)). BLAST programs are accessible through the U.S. National Library of Medicine, for example, on the world wide web at the URL "ncbi.nlm.nih.gov".

An agent that is suspected of having the ability to modulate BK channel activity and, therefore, circadian regulated locomotor activity or the sleep/wake cycle (referred to generally herein as a "test agent"), can have any chemical structure. As such, the agent can be a polynucleotide, a peptide, a peptidomimetic, a peptoid, a small organic molecule, and the like. Furthermore, the screening methods of the invention are adaptable to high throughput formats and, therefore, conveniently allow the examination of libraries of test agents, including combinatorial libraries, which can be randomized, biased, or variegated (see, for example, U.S. Pat. No. 5,837,500, which is incorporated herein by reference). Methods for preparing a combinatorial library of molecules that can be tested for a desired activity are well known in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U.S. Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith, Science 249:386, 1992; Markland et al., Gene 109:13-19, 1991; each of which is incorporated herein by reference); a peptide library (U.S. Pat. No. 5,264,563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83, 1995; a nucleic acid library (O'Connell et al., Proc. Natl. Acad. Sci., USA 93:5883, 1996; Tuerk and Gold, Science 249:505, 1990; Gold et al., Ann. Rev. Biochem. 64:763, 1995, each of which is incorporated herein by reference); an oligosaccharide library (York et al., Carb. Res., 285:99, 1996; Liang et al., Science, 274:1520, 1996; Ding et al., Adv. Expt. Med. Biol., 376:261-269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett., 399:232, 1996, which is incorporated herein by reference); a glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol., 130:567, 1995, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem., 37:1385, 1994; Ecker and Crooke, BioTechnology, 13:351, 1995; each of which is incorporated herein by reference).

An agent suspected of having the ability to modulate BK channel activity and, therefore, circadian regulated locomotor activity and/or a sleep/wake cycle in a subject can be a peptide. As used herein, the term "peptide" refers to a polymer comprising two or more amino acid residues or amino acid analogs that are covalently linked by a peptide bond, which can be a modified peptide bond. For example, a peptide test agent can contain one or more D-amino acids, or one or more amino acid analogs, for example, an amino acid that has been derivatized or otherwise modified at its reactive side chain. Similarly, one or more peptide bonds in the peptide can be modified, or the reactive group at the amino terminus or the carboxy terminus or both can be modified. Such peptides can be modified, for example, to have improved stability to a protease, an oxidizing agent or other reactive material the peptide can encounter in a biological environment, and, therefore, can be particularly useful for administration to a subject, which can be a test subject or a subject to be treated according to a method of the invention. Conversely, if desired, peptides can be designed to have decreased stability in a biological environment, for example, by including protease sensitive sites, such that the period of time the peptide is active in the environment is reduced.

A test agent also can be a polynucleotide. As used herein, the term "polynucleotide" means a polymer of two or more deoxyribonucleotides or ribonucleotides, or analogs thereof, that are linked together by a phosphodiester or other bond. As such, the terms include RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Although the term "polynucleotide" is used herein to include naturally occurring nucleic acid molecules such as those encoding BK channel polypeptides, polynucleotides useful as test agents generally are non-naturally occurring molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). Polynucleotides can be particularly useful as agents that modulate BK channel activity and, therefore, circadian regulated locomotor activity or sleep/wake cycle because nucleic acid molecules having binding specificity for cellular targets, including cellular polypeptides, exist naturally, and because synthetic molecules having such specificity can be readily prepared and identified (see, for example, U.S. Pat. No. 5,750,342, which is incorporated herein by reference).

The nucleotides comprising a polynucleotide can be naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose; or nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220 (1994); Jellinek et al., Biochemistry 34:11363 (1995); Pagratis et al., Nature Biotechnol. 15:68 (1997), each of which is incorporated herein by reference). The covalent bond linking the nucleotides of a polynucleotide can be a phosphodiester bond, or any of numerous other covalent bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tam et al., Nucl. Acids Res. 22:977 (1994); Ecker and Crooke, BioTechnology 13:351360 (1995), each of which is incorporated herein by reference). The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified molecules can be less susceptible to degradation.

A polynucleotide containing naturally occurring nucleotides and phosphodiester bonds, can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide containing nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al., supra, 1995).

An agent that modulates circadian regulated locomotor activity and/or the sleep/wake cycle can act by increasing BK channel activity or by decreasing BK channel activity, including by increasing or decreasing the activity of a mutant BK channel. Increased or decreased BK channel activity due to contact with a test agent can be examined using any of various methods known in the art for measuring potassium channel activity (see, for example, Meth. Enzymol.: Ion Channels (eds. Abelson et al., Academic Press 1998), which is incorporated herein by reference). For example, BK channel activity can be examined by making electrophysiological recordings such as by performing patch-clamp electrophysiologic analysis of cells following stable or transient transfection of cDNA molecules encoding slo and, if desired, slob or orthologs thereof, or voltage-clamp recording of Xenopus oocytes upon mRNA injection, cRNA, or cDNA injection. Extracellular or intracellular recordings of transfected cells can be obtained.

Extracellular voltage recording requires measurements of small biological potentials, often less than a millivolt in amplitude. The Axoclamp-2B, GeneClamp 500B and MultiClamp 700A microelectrode amplifiers are useful for such experiments (Axon Instruments, Inc.; Union City Calif.). Voltage clamp allows measurement of membrane current by monitoring the membrane voltage and injecting current to attain and maintain the desired voltage. As such, a voltage-clamp amplifier is selected based on its ability to measure voltage, and passes current in order to regulate the cellular voltage. Patch-clamp analysis utilizes a blunt pipette to isolate a patch of membrane. Patch-clamp recording can measure the individual ion channel currents that contribute to whole cell currents, and is compatible with current-clamp and voltage-clamp recording modes (see Axon Instruments, Inc., web site on the world wide web at URL "axon.com", information under "neurosciences" and "cellular neurosciences product lines"). In whole cell patch-clamping, the patch of membrane beneath the pipette is ruptured or otherwise made permeable such that currents passing through an entire cell membrane are recorded. This method is equivalent to intracellular recording with sharp microelectrodes, but has the advantage that it can be applied to cells that are very tiny or flat and would otherwise be very difficult to impale. The magnitude of the transmembrane current varies greatly between cell types. As such, the use of two electrodes, one for passing current and one for measuring voltage, is best for clamping large cells with large currents (Id.). Voltage clamp amplifiers such as the Axoclamp-2B amplifier and GeneClamp 500B amplifier are particularly useful for measurements using the Two-Electrode Voltage-Clamp (TEVC) mode (Axon Instruments, Inc.).

Current-clamp amplifiers are designed to control the current and measure the corresponding membrane voltage. It is common to pass current to stimulate a cell or modify its resting potential during intracellular voltage recording. The Axoclamp-2B amplifier, GeneClamp 500B amplifier, and MultiClamp 700A amplifier (Axon Instruments, Inc.) can pass current while in voltage-sensing (i.e., current-clamp) mode. The Axoclamp-2B amplifier also allows for "discontinuous" recording modes, applicable to both voltage clamp and current clamp. In this mode the instrument divides its time in passing current and recording voltage. The advantage of this mode is that the recording is free from the usual error due to the voltage drop across the electrode resistance, and can be used with a conventional intracellular microelectrode. Ion-selective electrodes, voltammetry and constant-voltage amperometry also can be used to measure levels and small changes in ion, neurotransmitter and hormone concentrations in tissues or in and near cells. These techniques require the ability to record small potentials and pass large currents. Ion-selective electrodes require differential input, low leakage current and high-impedance voltage following. The electrochemical techniques of voltammetry and constant-voltage amperometry are used to measure fast changes in neurotransmitter concentrations, and require a voltage-clamp amplifier with a command voltage range extended to .+-.1V (Axon Instruments, Inc.).

Detection of BK channel activity in native systems or in recombinant expression systems also can be examined using fluorescent dyes sensitive to membrane potential or intracellular ions (including pH). The Voltage Ion Probe Reader II system (VIPR II.TM. system) applies fluorescence resonance energy transfer (FRET) technology to ion channel analysis (Aurora Biosciences Corp., San Diego Calif.; see web site on the world wide web at the URL "aurorabio.com", information under "Aurora platforms" and "ion channel technology"). FRET is a distance-dependent interaction between the electronic excited states of two dye molecules, and is useful for investigating biological events that produce changes in molecular proximity, including, for example, FRET between a membrane-bound donor molecule and a mobile, voltage-sensitive, acceptor molecule to detect membrane potential ("Voltage Sensor Probe Technology"; Aurora Biosciences Corp.). The VIPR II.TM. system can be performed in a 96 well format or 384 well format and, therefore, is particularly suitable for high throughput screening assays, allowing for throughput of up to 40,000 samples per day under temperature controlled conditions. The system allows for dual emission fluorescence kinetic reading in real time, and includes data collection and analysis software. The system can screen potassium and calcium gated ion channels, reads approximately 5 mV changes in membrane potential in milliseconds, and allows for single cell detection.

Aurora Biosciences Corp. also provides "Voltage Sensor Probes" technology, which combine rapid response and high sensitivity for reliable detection of changes in membrane voltage induced by modulation of ion channels (see web site on the world wide web at the URL "aurorabio.com", information under "bioassay technologies" and "voltage sensitive probes"; see, also, "fluorescent probes"). Voltage Sensor Probes technology uses two fluorescent molecules, including oxonol, which is a highly fluorescent, negatively charged, hydrophobic ion that "senses" the transmembrane electrical potential, and coumarin lipid, which binds specifically to one face of the plasma membrane and functions as a FRET donor to the voltage-sensing oxonol acceptor molecule. In response to changes in membrane potential, oxonol can rapidly redistribute between two binding sites on opposite sides of the plasma membrane. When oxonol moves to the intracellular plasma membrane binding site upon depolarization, FRET is decreased and results in an increase in the donor fluorescence and a decrease in the oxonol emission (Id.).

Another fluorimetric system developed to measure channel activity is the fluorimetric imaging plate reader (FLIPR.TM.) system (Molecular Devices Corp.; Sunnyvale Calif.; see web site on world wide web at URL "moleculardevices.com", search "FLIPR calcium assay"). The FLIPR.TM. system conveniently can be performed in a 384 well high throughput format (FLIPR.sup.384) using a minimal sample volume. The FLIPR.sup.384 system can monitor intracellular calcium, membrane potential, intracellular pH, and intracellular sodium from cells of a population in real time, providing maximum versatility and the ability to identify a potential hit seconds after it is added to the cell plate. Real-time, kinetic data also provides additional pharmacological information for ranking relative potencies of drugs, and gives information on the kinetics of the drug-receptor interaction (Id.).

BK channel activity also can be examined using an ion flux assay, for example, a rubidium ion efflux assay, which provides a functional analysis. Rubidium ion is similar in size and charge to potassium ion and confers similar permeability rates within the cell. BK channel activity can be determined by quantifying rubidium ion levels in cell lysate and supernatant fractions, wherein rubidium ion concentration in the fractions is directly related to channel efflux. Rubidium ion concentration can be determined using flame atomic absorption spectroscopy method, which can be automated using, for example, an ICR 8000 system (Aurora Biomed, Inc.; Vancouver BC; see, also, Aliphitiras et al., Soc. Biomol. Screening, 7th Ann. Conf., Poster Session 5, #5004, which is incorporated herein by reference).

Calcium ion flux also can be measured using dyes such as fura-2, indo-1, or derivatives thereof, which are UV light-excitable, ratiometric calcium ion indicators (Molecular Probes, Inc.; Eugene Oreg.). Fura-2, for example, is useful for ratiometric imaging, and exhibits an absorption shift that can be observed by scanning the excitation spectrum between 300 nm and 400 nm, while monitoring the emission at approximately 510 nm. In comparison, indo-1 is useful flow cytometry analysis. The emission maximum of indo-1 shifts from approximately 475 nm in calcium ion-free medium to about 400 nm when the dye is saturated with calcium ion. The sodium and potassium salts of fura-2 and the potassium salt of indo-1 are cell-impermeant probes that can be delivered into cells by microinjection or using an influx pinocytic cell-loading reagent (Molecular Probes, Inc.; see web site on world wide web at URL "molecularprobes.com", information under "Handbook" and "chapter 20"). Quin-2 is another calcium ion indicator that has lower absorptivity and quantum yield values than the fura-2 and indo-1 and, therefore, requires higher loading concentrations, which can buffer intracellular calcium ion transients (Id.).

BK channel activity also can be examined using ion chelators or other extracellular or intracellular reagents that exhibit a change in physico-chemical properties upon potassium binding, or by detecting changes in intracellular or extracellular pH levels (see, for example, U.S. Pat. No. 6,150,176; U.S. Pat. No. 6,140,132, each of which is incorporated herein by reference). In addition, changes in BK channel activity can be detected by examining changes in the expression of genes that are regulated by such changes, or by measuring the activity of calcium channels that are co-expressed with the BK channels and sensitive to membrane potential.

A change in BK channel activity also can be detected by examining the expression of a reporter gene that is operatively linked to a gene regulatory element that is responsive to the change in BK channel activity. For example, glucagon contains a calcium response element, which regulates glucagon expression in response to calcium ion concentration (Furstenau et al., J. Biol. Chem. 274:5851, 1999, which is incorporated herein by reference). As such, the calcium response element can be operatively linked to a reporter gene, which, upon introduction into a cell being examined according to a method of the invention, provides a means to detect changes in intracellular calcium ion due to an effect of a test agent on BK channel activity. Other gene regulatory elements that are responsive to changes in calcium ion include, for example, the cAMP response element and the serum response element (see, for example, Ginty, Neuron 18:183, 1997, which is incorporated herein by reference).

Reporter genes that can be operatively linked to a desired regulatory element are well known in the art and include, for example, a .beta.-lactamase, chloramphenicol acetyltransferase, adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin-B phosphotransferase, thymidine kinase, .beta.-galactosidase, luciferase and xanthine guanine phosphoribosyltransferase polypeptide. Similarly, methods of detecting expression of such reporter genes are well known and include, for example, methods of detecting a colorimetric, luminescent, chemiluminescent, fluorescent, or enzymatic activity due to expression of the reporter polypeptide.

As used herein, the term "operatively linked" means that two or more molecules are positioned with respect to each other such that they act as a single unit and effect a function attributable to one or both molecules or a combination thereof. For example, a polynucleotide sequence encoding a reporter polypeptide or the like can be operatively linked to a regulatory element such as a calcium response element, in which case the regulatory element confers calcium inducible expression on the reporter similarly to the way in which the regulatory element would effect, for example, expression of glucagon in a pancreatic islet cell. A first polynucleotide coding sequence also can be operatively linked to a second (or more) coding sequence such that a chimeric polypeptide can be expressed from the operatively linked coding sequences. The chimeric polypeptide can be a fusion polypeptide, in which the two (or more) encoded peptides are translated into a single polypeptide, i.e., are covalently bound through a peptide bond; or can be translated as two discrete peptides that, upon translation, can operatively associate with each other to form a stable complex. For example, the fusion protein can comprise a fluorescent protein, which can be useful for constructing chimeric proteins for FRET analysis.

A change in BK channel activity also can be detected using a physical method such as Fourier transform infrared analysis, atomic force microscopy (Chen and Hansma, J. Struct. Biol. 131:44, 2000; Oesterhelt et al., Science, 143, 2000; Stolz et al., J. Struct. Biol. 131:171, 2000; Obregon et al., Biophys. J. 79:202, 2000, each of which is incorporated herein by reference), Raman spectroscopy, and the like, or by detecting a conformational change of the BK channel or a change in protein-protein interaction such as between the BK channel and a BK channel binding protein using a method such as FRET (U.S. Pat. No. 6,342,379; U.S. Pat. No. 5,661,035, each of which is incorporated herein by reference), B-RET, FIDA, FP, FCS. Such methods can be utilized for detecting changes in BK channel in a cell or changes in a substantially purified BK channel in vitro.

Various drugs can act as potassium channel antagonists and, therefore, can be useful for confirming the accuracy and validity of a test system comprising a BK channel and as controls that can be run in parallel with test agents. Such drugs include glyburide (1-{{{p-2-(5-chloro-o-anisamido)ethyl}phenyl}-sulfonyl}-3-cyclohexylurea)- , glipizide (1-cyclohexyl-3-{{{p-(2-(5-methylpyrazinecarboxamido)ethyl}phenyl}sulfony- l}urea) and tolbutamide (1-butyl-3-(p-methylbenzenesulfonyl)urea), which are used as anti-diabetic agents, and other antagonists that are used as Class III anti-arrhythmic agents and to treat acute myocardial infarctions in humans. A number of naturally occurring toxins that block potassium channels, including apamin, IBX, CbTX, margatoxin, noxiustoxin, kaliotoxin, dendrotoxin(s), mast cell degranuating peptide, and .beta.-bungarotoxin, also can be used for this purpose.

A screening assay of the invention provides a means to identify agent that can modulate circadian regulated locomotor activity and/or the sleep/wake cycle of an individual. As used herein, the term "sleep/wake cycle" refers to a rhythmic pattern, in which sleep onset leads to a period of sleep, followed by awakening from sleep, and a period of wakefulness, to be followed again by sleep onset, and so on. A normal sleep/wake cycle generally repeats in a circadian rhythm over a period of about 24 hours, and is linked to the length of day and night, i.e., the light/dark cycle. Reference herein to a "normal" or "typical" sleep/wake cycle means the sleep/wake cycle that is characteristic of a population of organisms in nature. For example, a normal sleep/wake cycle in humans includes sleep onset occurring about 4 to 5 hours after sunset, followed by a period of sleep that ends with awakening about 1 to 2 hours after sunrise (see, for example, Young and Kay, Nat. Rev. Genet. 2:702, 2001, which is incorporated herein by reference). In comparison, a normal sleep/wake cycle for nocturnal organisms is characterized by sleep onset occurring at or about sunrise or shortly thereafter.

Although actual sleep/wake cycles can vary substantially among individuals of a population, observation over a period of time and of specific individual or of a representative population of individuals and routine statistical analyses can be used to determine a sleep/wake cycle characteristic of the specific individual (prior to and/or after treatment with a test agent) or of the population, which can be a population of otherwise healthy individuals or a population of individuals suffering from the same disorder, for example, insomnia. It is recognized that in most populations, including humans, there will be a wide range of times "normal" individuals awaken or drowse to sleep. Nevertheless, any population of individuals will exhibit a normal distribution of such times and, therefore, an mean and standard deviation can be determined. For example, humans, on average, sleep for about eight hours and are awake for about sixteen hours.

Circadian rhythms are nearly ubiquitous in nature, occurring in prokaryotes and eukaryotes. The processes under circadian control are equally diverse, ranging from human sleep/wake cycles to cell division in photosynthetic bacteria. The hallmark of these roughly 24 hour rhythms is their persistence under constant environmental conditions. This persistence is effected by the circadian clock, which is an internal biochemical oscillator. The circadian clock allows an organism to anticipate daily changes in the environment such as the onset of dawn and dusk, thereby providing the organism with an adaptive advantage (Yan et al., Proc. Natl. Acad. Sci., USA 95:8660, 1998). As such, the term "circadian regulated locomotor activity" is used herein to refer to rhythmic activity that is anticipatory of a daily change in the environment, particularly rhythmic activity that is anticipatory of the onset of dawn and dusk. In addition, the circadian clock regulates other rhythmic activities, including, for example, hormonal rhythms, blood pressure rhythms, body temperature rhythms, cholesterol production, and heme production. A normal pattern for a circadian regulated locomotor activity or other circadian regulated rhythmic activity can be determined by observing individuals in a population and analyzing the results using statistical methods, as discussed below with respect to determining a normal sleep/wake cycle. Such methods provide a standard value, from which individuals exhibiting an arrhythmia in the locomotor activity can be identified (see, also, Example 1).

In a screening assay of the invention, an agent suspected of having the ability to modulate circadian regulated locomotor activity and/or the sleep/wake cycle is examined initially for the ability to modulate BK channel activity, then agents that are identified as having the ability to modulate BK channel activity are administered to test subjects to identify those agents that also modulate circadian regulated locomotor activity, the sleep/wake cycle, or both. The test subject can be any organism that expresses wild type or mutant BK channels in nerve cells and muscle cells, including an organism that has been genetically modified to express such BK channels, for example, a transgenic non-human organism. Generally, the test subject contains cells that express substantially the same BK channels as were used in the initial test system to identify agents that modify BK channel activity. However, the test subject also can contain cells that express a homolog, ortholog or paralog of the BK channel used in the initial test system, or a mutant or other variant of the BK channel. In addition, the screening method can include one or more control subjects, which, for example, contain cells that express a BK channel that is not modulated by the test agent, thus providing a means to confirm the specificity of an agent that is found to modulate the sleep/wake cycle or circadian regulated locomotor activity of a test subject.

A test subject is selected, in part, based on the characteristics desired of the test agent being examined. For example, if test agents are being screened to identify those that can shift sleep and awakening by a desired time (e.g., about four, six, eight or twelve hours), the test subjects can be organisms that exhibit a normal sleep/wake cycle. In this respect, it should be recognized that many organisms, including experimental organisms such as mice, are primarily nocturnal. As such, while in humans, a "normal" sleep/wake cycle includes awakening about dawn (CT0) and drowsing to sleep after dusk (CT12), for example, at about CT16 (assuming an average of 8 hours sleep), the cycle can be different in an experimental organism that is used as a test subject for identifying an agent that would be useful in humans. Nevertheless, the effectiveness that an agent being tested in an experimental animal can have in a human can be determined by accounting for these differences. Where a test agent is being examined, for example, to treat insomnia, the test subject can be selected based on having symptoms of insomnia, or can be placed in conditions that are not conducive to sleep, and the ability of an agent that modulates BK channel activity to allow sleep to begin at a normal time can be examined. Where a test agent is being examined for an ability to induce circadian regulated locomotor activity, the test subject is selected based on a lack of such regulated activity, for example, a test subject exhibiting anxiety or hyperkinetic activity, or a subject exhibiting a circadian related sleep disorder such as familial advance phase sleep disorder or familial delayed phase sleep disorder (see, for example, Sleep 22:616-623, 1999).

An agent that modulates the sleep/wake cycle can be identified by detecting a change in the sleep/wake pattern of the subject, either in comparison to the sleep/wake pattern in the subject prior to administration of the test agent, or in comparison to a sleep/wake pattern characteristic of a normal population comprising the subject. The agent can be administered to the test subject (or control subject) in any convenient manner, including, for example, orally or by injection (see, also, below). Using such a screening assay as disclosed herein, agents that modulate the sleep/wake cycle or circadian regulated locomotor activity in a subject by modulating BK channel activity can be identified. Accordingly, the present invention provides agents identified by a screening assay. Such agents can be useful as medicaments for treating a subject having, for example, a disorder affecting the sleep/wake cycle.

The present invention also provides a method of modulating the sleep/wake cycle in a subject by administering an agent that modulates the activity of a clock regulated gene product, for example, BK channel activity, to the subject. The subject can be any subject in which it is desired to modulate the sleep/wake cycle. Generally, a subject to be treated is one suffering from an acute or chronic sleep disorder, wherein administration of the agent modulates the sleep/wake cycle of the subject so as to more closely approximate the sleep/wake cycle of a normal population to which the subject belongs. A sleep disorder amenable to treatment according to a method of the invention is characterized, in part, by an inability of the subject to establish a regular pattern of sleep, for example, insomnia or narcolepsy. A subject to be treated also can be one wishing to change his or her otherwise normal sleep/wake cycle. For example, a person preparing to travel to a different time zone, particularly a time zone that is at least about four hours or six hours different from that in which the person is leaving, can take an agent that delays (or advances) the current sleep/wake cycle a sufficient amount such that the person can avoid jet lag. Similarly, a person that works a night shift can benefit from a change in an otherwise normal sleep/wake cycle to one that accommodates his or her work schedule. As such, a method of the invention can provide advantages to the general public, including, for example, decreased risk of injuries due to tiredness during a night shift, or increased productivity of business person traveling to a distant time zone.

The present invention also provides a method of modulating circadian regulated locomotor activity in a subject by administering an agent that modulates BK channel activity to the subject. The subject can be, for example, a person suffering from an anxiety or hyperkinetic disorder, wherein administration of agent that attenuates the locomotor activity associated with the disorder provides a rhythmic decreased locomotor activity. Such a rhythmic decreased locomotor activity can be timed, for example, such that the subject can remain in a more "relaxed state" during work or school hours. A method of the invention also can be useful for treating a herd of farm animals, such that individual animals in the herd are not overly disruptive or such that the herd, in general, is more amenable to handling during desired times.

The agent can be administered to a subject by any convenient means, including, for example, orally in the form of a tablet or a capsule, or as a component of food or water to which the subject has access. The agent also can be administered, for example, via a pump or can be formulated in a time-released form, thus providing a means to maintain the agent at a desired level over a period of time. A time-released form of the agent can be contained, for example, in a matrix, which can be administered to a subject intradermally, subcutaneously, or intramuscularly.

Generally, for administration to a subject, the agent is formulated in a composition suitable for administration to the subject. As such, the present invention also provides compositions containing an agent, which modulates BK channel activity and further modulates the sleep/wake cycle or circadian regulated locomotor activity, in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the agent and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art.

The agent can be incorporated within an encapsulating material such as into an oil-in-water emulsion, a microemulsion, micelle, mixed micelle, liposome, microsphere or other polymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981), each of which is incorporated herein by reference). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. "Stealth" liposomes (see, for example, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of such encapsulating materials, as are cationic liposomes, which can be modified with specific receptors or ligands, for example, to target muscle tissue or brain (Morishita et al., J. Clin. Invest., 91:2580-2585 (1993), which is incorporated herein by reference).

The route of administration of a composition containing an agent that modulates the sleep/wake cycle or circadian regulated locomotor activity will depend, in part, on the chemical structure of the molecule. Polypeptides and polynucleotides, for example, are not particularly useful when administered orally because they can be degraded in the digestive tract. However, methods for chemically modifying polypeptides, for example, to render them less susceptible to degradation by endogenous proteases or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., supra, 1995; Ecker and Crook, supra, 1995). In addition, a polypeptide agent can be prepared using D-amino acids, or can contain one or more domains based on peptidomimetics, which are organic molecules that mimic the structure of peptide domain; or based on a peptoid such as a vinylogous peptoid.

A composition as disclosed herein can be administered to an individual by various routes, including, for example, topically, orally or parenterally, such as intravenously, intramuscularly, subdermally, or subcutaneously, or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively, or using a nasal spray or inhalant, in which case one component of the composition is an appropriate propellant. Preferably, the composition is administered orally, for example, as a component of a food or beverage, or is administered in a time released formulation. Where a group of subjects is to be treated, for example, a herd of farm animals, the composition can be incorporated in the livestock food or water.

One skilled in the art would know that the amount of the composition to administer to a subject depends on many factors including the age and general health of the subject as well as the route of administration. In view of these factors, the skilled artisan would adjust the particular dose as necessary. Appropriate amounts having the desired efficacy can be determined using routine methods. When humans are to be treated, the formulation of the composition and the routes and frequency of administration are determined, initially, using Phase I and Phase II clinical trials.

A composition for oral administration can be formulated, for example, as a tablet, or a solution or suspension form; or can comprise an admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications, and can be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use. The carriers, in addition to those described above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see, for example, U.S. Pat. No. 5,314,695). Additional formulations can be determined based on the particular subjects to be treated.
 

Claim 1 of 33 Claims

1. A method of identifying an agent that can modulate the sleep/wake cycle in a subject, the method comprising: contacting a test system comprising a BK channel and a cognate BK channel binding protein with an agent suspected of having the ability to modulate the sleep/wake cycle in the subject, wherein the BK channel comprises a Drosophila slowpoke (slo) polypeptide, or an ortholog thereof, wherein the ortholog is human slo, murine slo, rat slo, or rabbit slo; detecting a change in: i) conformation of the BK channel or protein-protein interaction between the BK channel and the BK channel binding protein, and ii) intracellular ions induced by the agent as compared to the same measurements taken in the absence of the agent, thereby identifying an agent that modulates BK channel activity; administering the agent that modulates BK channel activity to a test subject; and thereafter detecting a change in the sleep/wake cycle of the test subject, thereby identifying an agent that can modulate the sleep/wake cycle in a subject.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.