Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-22
2002-08-27
Clark, Deborah J. R. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S320100, C435S325000, C435S455000, C530S350000
Reexamination Certificate
active
06441156
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to calcium channel compositions and methods of making and using same. In particular, the invention relates to calcium channel alpha2delta (&agr;
2
&dgr;) subunits and nucleic acid sequences encoding them. These compositions are useful in methods for identifying compounds that modulate the activity of calcium channels and for identifying compounds as therapeutic for disease.
BACKGROUND OF THE INVENTION
Calcium channels are present in various tissues, have a central role in regulating intracellular calcium ion concentrations, and are implicated in several vital processes in animals (e.g., neurotransmitter release, muscle contraction, pacemaker activity, secretion of hormones and other substances, etc.). Thus, changes in calcium influx into cells which are mediated through calcium channels have been implicated in various human diseases such as disorders of the central nervous system and cardiovascular disease.
For example, changes to calcium influx into neuronal cells may be implicated in conditions such as epilepsy, stroke, brain trauma, Alzheimer's disease, multiinfarct dementia, other classes of dementia, Korsakoff's disease, neuropathy caused by a viral infection of the brain or spinal cord (e.g., human immunodeficiency viruses, etc.), amyotrophic lateral sclerosis, convulsions, seizures, Huntington's disease, amnesia, or damage to the nervous system resulting from reduced oxygen supply, poison or other toxic substances (See e.g., Goldin et al., U.S. Pat. No. 5,312,928).
Additionally, changes to calcium influx into cardiovascular cells may be implicated in conditions such as cardiac arrhythmia, angina pectoris, hypoxic damage to the cardiovascular system, ischemic damage to the cardiovascular system, myocardial infarction, and congestive heart failure (Goldin et al., supra). Other pathological conditions associated with elevated intracellular free calcium levels include muscular dystrophy and hypertension (Steinhardt et al., U.S. Pat. No. 5,559,004). While there has been limited success in expressing DNA encoding rabbit and rat calcium channel subunits, little is known about human calcium channel structure, function and gene expression. Additionally, there is limited knowledge in the art of the role of calcium channel types in cell growth control and abnormalities of calcium channels leading to cancer development.
In addition to the implication of calcium channels in animal (including human) diseases, a number of compounds which are currently used for treating various cardiovascular diseases in animals (including humans) are believed to exert their beneficial effects by modulating the functions of voltage-dependent calcium channels present in cells, such as cardiac cells and vascular smooth muscle cells. Nonetheless, there is a paucity of understanding of the pharmacology of compounds which interact with calcium channels. This paucity of understanding, together with the limited knowledge in the art of the human calcium channel types, the molecular nature of the human calcium channel subtypes, and the limited availability of pure preparations of specific calcium channel subtypes to use for evaluating the efficacy of calcium channel-modulating compounds has hampered the rational testing and screening of compounds that interact with the specific subtypes of human calcium channels to have desired therapeutic effects.
SUMMARY OF THE INVENTION
The invention provides calcium channel alpha2delta (&agr;
2
&dgr;) subunits, as well as amino and nucleic acids encoding them. In one embodiment, the invention provides a substantially purified nucleic acid sequence consisting of at least a portion of a nucleotide sequence selected from the group consisting of (a) SEQ ID NO:1, the complement thereof, variants thereof, and homologs thereof, (b) SEQ ID NO:3, the complement thereof, variants thereof, and homologs thereof, and (c) SEQ ID NO:5, the complement thereof, variants thereof, and homologs thereof. In a preferred embodiment, the nucleic acid sequence encodes at least a portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof. In a more preferred embodiment, the nucleic acid sequence is double-stranded. In an alternative more preferred embodiment, the nucleic acid sequence is single-stranded.
Without intending to limit the nucleic acid sequences of the invention to any particular type of encoded protein, in an alternative preferred embodiment, the nucleic acid sequence encodes a fusion protein. While it is not contemplated that the invention be limited to the type or nature of fusion partner in the fusion protein, in a more preferred embodiment, the fusion protein comprises a polypeptide selected from the group consisting of chloramphenicol acetyltransferase, luciferase, beta-galactosidase, green fluorescent protein, Myc protein, protein A, glutathione-S-transferase, FLAG tag, and polyhistidine.
In another alternative preferred embodiment, the nucleic acid sequence is contained on a recombinant expression vector. In a more preferred embodiment, the expression vector is contained within a host cell. While it is not contemplated that the invention be limited to the type of host cell, in a yet more preferred embodiment, the host cell is eukaryotic. In an even more preferred embodiment, the eukaryotic cell is selected from the group consisting of cancer cells and amphibian oocytes.
Also provided by the invention is a substantially purified amino acid sequence comprising at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof. In a preferred embodiment, the portion is part of a fusion protein. While not intending to limit the invention to any particular type or nature of fusion protein, in a more preferred embodiment, the fusion protein comprises a polypeptide selected from the group consisting of chloramphenicol acetyltransferase, luciferase, beta-galactosidase, protein A, glutathione-S-transferase, FlAG tag, and polyhistidine.
The invention further provides substantially purified amino acid sequences encoded by at least a portion of a nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 1, the complement thereof, variants thereof, and homologs thereof, (b) SEQ ID NO:3, the complement thereof, variants thereof, and homologs thereof, and (c) SEQ ID NO:5, the complement thereof, variants thereof, and homologs thereof.
The invention additionally provides methods for detecting presence of a nucleic acid sequence encoding at least a portion of a calcium channel protein, comprising: a) providing: i) a sample suspected of containing the nucleic acid sequence; and ii) at least a portion of a nucleotide sequence selected from the group consisting of (1) SEQ ID NO: 1, the complement thereof, variants thereof, and homologs thereof, (2) SEQ ID NO:3, the complement thereof, variants thereof, and homologs thereof, and (3) SEQ ID NO:5, the complement thereof, variants thereof, and homologs thereof, b) combining the sample with at least a portion of the nucleotide sequence under conditions such that the nucleic acid hybridizes with at least a portion of the nucleotide sequence; and c) detecting the hybridization. Although it is not contemplated that the invention be limited to the level of stringency of hybridization, in one preferred embodiment, the hybridization is under conditions of low stringency. In another preferred embodiment, the hybridization is under conditions of high stringency. Furthermore, it is contemplated that the invention will also be used in various assays to detect mRNA using these sequences (i.e., SEQ ID NOS:1, 3, and 5) including, but not limited to microassays.
The invention also provides methods for producing at least a portion of a calcium channel protein, comprising: a) providing: i) a recombinant expression vector comprising a nucleic acid seque
Duh Fuh-Mei
Gao Boning
Latif Farida
Lerman Michael Isaac
Minna John Dorrance
Clark Deborah J. R.
Medlen & Carroll LLP
Paras, Jr. Peter
The United States of America as represented by the Department of
LandOfFree
Calcium channel compositions and methods of use thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Calcium channel compositions and methods of use thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Calcium channel compositions and methods of use thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2885275