Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
2000-05-25
2004-02-17
Kunz, Gary (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C435S069100, C435S071100, C435S471000, C435S320100
Reexamination Certificate
active
06693172
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a novel methods for measuring cellular ion channel transmission and methods and compositions useful in the identification of ligand gated ion channel agonists and modulators.
BACKGROUND OF THE INVENTION
Ion Channels
Ion channel proteins form hydrophilic pores that extend across the cellular lipid bilayer; when these pores are open, they allow specific molecules (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane.
Channel proteins which are concerned specifically with inorganic ion transport are referred to as ion channels, and include ion channels for sodium, potassium, calcium, and chloride ions. Ion channels which open in response to a change in the voltage across the membrane are referred to as voltage gated ion channels (or voltage-dependent ion channels). Ion channels which open in response to the binding of a ligand to the channel protein are referred to as ligand gated ion channels.
The present invention describes new ion channels and provides methods and compositions suitable for high throughput screening of ion channels.
DESCRIPTION OF THE INVENTION
Voltage Gated Ion Channels
Voltage Gated Sodium Channel
Voltage gated ion channels are a class of channel proteins that play a major role in cellular electrical excitability. In the majority of excitable tissues, the early depolarization phase of action potentials is mediated by a sodium current via voltage-dependent sodium channels (also known as voltage-gated sodium channels or VGSCs). The sodium channel is one of the most thoroughly characterized of the voltage gated channels. The primary structures of many sodium channels from a variety of tissues (brain, skeletal muscle and cardiac muscle) and organisms (jellyfish, squid, eel, rat, human) have been identified, and their amino acid sequences show individual regions which are highly conserved over evolution, indicating that voltage-dependent sodium channels belong to a large superfamily of evolutionarily related proteins. All published polypeptide complexes of VGSCs have in common a large, about 260 kDa glycoprotein (the pore forming subunit) which is called the alpha subunit (Agnew et al. 1978; Agnew et al. 1980; Catterall 1986; Catterall 1992). Additional lower molecular weight polypeptides, the beta-subunits, have been found to be associated with sodium channels from mammalian muscle (Kraner et al. 1985; Tanaka et al. 1983) and brain (Hartshorne and Catterall 1984). The large, pore-forming alpha subunit is sufficient for all known functions of VGSCs (Catterall 1992) while the beta subunits modulate some of the functions of the alpha subunit (Catterall 1992).
Voltage Gated Potassium Channels
Voltage-gated potassium channels make up a large molecular family of integral membrane proteins that are fundamentally involved in the generation of bioelectric signals such as nerve impulses. These proteins span the cell membrane, forming potassium-selective pores that are rapidly switched open or closed by changes in membrane voltage. Several chemical entities have been discovered to be potent and specific openers of vascular potassium K+ channels. These include cromakalim and its derivatives and RP 52891. This mechanism is also shared, at least partially, by drugs such as minoxidil, diazoxide, pinacidil and nicorandil. The opening of plasmalemmal K+ channels produces loss of cytosolic K+. This effect results in cellular hyperpolarization and functional vasorelaxation. In normotensive or hypertensive rats, K+ channel activators decrease aortic blood pressure (by producing a directly mediated fall in systemic vascular resistance) and reflexively increase heart rate. K+ channel openers produce selective coronary vasodilatation and afford functional and biochemical protection to the ischemic myocardium.
The structure of a typical voltage-gated potassium channel protein is known to be comprised of six membrane spanning domains in each subunit, each of which is regulated by changes in membrane potential. B. Hille. “Ionic Channels of Excitable Membranes”(Sinauer, Sunderland, Mass., 1992). Voltage-gated potassium channels sense changes in membrane potential and move potassium ions in response to this alteration in the cell membrane potential. Molecular cloning studies on potassium channel proteins has yielded information primarily for members of the voltage-gated family of potassium channels. Various genes encoding these voltage-gated family of potassium channel proteins have been cloned using Drosophila genes derived from both the Shaker, Shaw and Shab loci; Wei, A. et. al., Science (1990) Vol. 248 pp. 599-603.
Voltage Gated Calcium Channels
Voltage-gated calcium channels are present in neurons, and in cardiac, smooth, and skeletal muscle and other excitable cells. These channels are known to be involved in membrane excitability, muscle contraction, and cellular secretion, such as in exocytotic synaptic transmission (McCleskey, et al.,1987). In neuronal cells, voltage-gated calcium channels have been classified by their electrophysiological as well as by their biochemical (binding) properties.
Calcium channels are generally classified according to their electrophysiological properties as Low-voltage-activated (LVA) or High-voltage-activated (HVA) channels. HVA channels are currently known to comprise at least three groups of channels, known as L-, N- and P-type channels (Nowycky, et al., 1985). These channels have been distinguished one from another structurally and electrophysiologically as well as biochemically on the basis of their pharmacology and ligand binding properties. Thus, dihydropyridines, diphenylalkylamines and piperidines bind to the alpha1 subunit of the L-type calcium channel and block a proportion of HVA calcium currents in neuronal tissue, which are termed L-type calcium currents.
N- or omega-type HVA calcium channels are distinguishable from other calcium channels by their sensitivity to omega conotoxins (omega conopeptides). Such channels are insensitive to dihydropyridine compounds, such as L-type calcium channel blockers nimodipine and nifedipine. (Sher and Clementi, 1991).
Ligand Gated Ion Channel Receptors
Ligand-gated ion channels provide a means for communication between cells of the central nervous system. These channels convert a signal (e.g., a chemical referred to as a neurotransmitter) that is released by one cell into an electrical signal that propagates along a target cell membrane. A variety of neurotransmitters and neurotransmitter receptors exist in the central and peripheral nervous systems. At the present time, numerous families of ligand-gated receptors have been identified and characterized on the basis of sequence identity these include nicotinic acetylcholine, glutamate, glycine, GABA A, 5-HT3, and the purinoceptors. These can be further characterized by whether the gated ion channel transmits cations or anions. Those which form cationic channels include, for example, excitatory nicotinic acetylcholine receptors (nAChRs), excitatory glutamate-activated receptors, the 5-HT3 serotonin receptor, and the purine receptor.
Those which form anionic channels include, for example, the inhibitory GABA and glycine-activated receptors. This discussion will confine itself to those ligand gated ion channel receptors which conduct cations.
5HT
3
Receptor
Molecular cloning has indicated that serotonin (5-hydroxytryptamine, also referred to as 5-HT) receptors belong to at least two protein superfamilies: G-protein-associated receptors and ligand-gated ion channel. The 5-HT
3
receptor belongs to the family of ligand-gated ion channels. As discussed below the 5-HT
3
receptor is primarily a sodium potassium ligand gated ion channel under physiologic conditions. The inflammatory and painproducing effects of serotonin are generally believed to be mediated via 5HT
3
receptors on peripheral sensory endings (Richardson, B. P., et al., 1985).
Nicotinic Receptors
The nicotinic acetylcholine receptors (nAChRs) are multisubunit proteins of
Berkenpas Mitchell B.
Groppi, Jr. Vincent E.
Wolfe Mark L.
Hamud Fozia
Kunz Gary
Pharmacia and Upjohn Company
Rehberg Edward F.
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