Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-02-17
1998-07-14
Achutamurthy, Ponnathapura
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, 514 1, G01N 2119, G01N 33566, A61K 4100, A61K 4748
Patent
active
057802424
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates generally to a bioassay technique to measure the interaction of molecules with ion channels and in particular, sodium channels. More specifically, the present invention relates to the use of circular dichroism to diagnose ion channel, i.e., potassium, calcium and preferably sodium channel disease or dysfunction and a method for screening ion channel active molecules. This invention further provides a novel means to treat cancer associated with diseased dysfunction of ion channels, and especially sodium channel thermal proteins.
BACKGROUND OF THE INVENTION
The ion channel, especially the sodium channel is a transmembrane protein responsible for the voltage-dependent modulation of the sodium ion permeability of excitable membranes and thus plays an essential role in generating action potentials. Hille, B., Ionic Channels of Excitable Membranes, Sinauer, Sunderland, Mass. (1984). Propagation of the action potential in nerve and muscle cells is generally thought to occur by transient changes in the permeability of the cell membrane to Na.sup.+, K.sup.+ or Ca.sup.++ ions via a specific channel. Two events can be distinguished in the passage of Na.sup.+, K.sup.+ or Ca.sup.++ through the channel: 1) selective filtering and 2) rapid increase in the permeability to Na.sup.+, K.sup.+ or Ca.sup.++ by a gating type of mechanism. Angelides, K. J. and T. J. Nuttov, J. Biol. Chem. 258:11858-11867 (1981). In addition, ion channels may be either voltage gated (e.g., Na.sup.+ and Ca.sub.2+ channels) implying that current is gated (or regulated) by membrane potential (voltage), or chemically gated (e.g., acetylcholine receptors and .gamma.-aminobutyric acid receptors) implying that current is gated primarily by binding of a chemical rather than by the membrane potential. Butterworth, J. F. and G. R. Strichartz, Anesthesiology 72:711-734 (1980).
Sodium channels have been isolated and purified by biochemical methods. See, Merksey, B. D., U.S. Pat. No. 4,895,807 (1990). Recently, sodium channels from the electric organ of the eel and rat brain have been cloned and sequenced. Kayano, et al. FEBS LETTERS 28: 181-184 (1988). The amino acid sequences of the Na.sup.+ channel found in eel electroplax has been deduced from its gene sequence. From this deduction, Kayano, et al. propose that the channel has large hydrophobic regions, probably in .alpha.-helical conformations that span the membrane, interspersed with hydrophilic regions to form a Na+ ion conducting "pore" of the channel. It is believed that the .alpha.-helical structures provide conformational flexibility for the sodium channel which is functionally responsible for the channels "open" and "close" gating mechanism. Oiki, et al., Proteins, 8:226-236 (1990).
The pharmacology of sodium channels has been extensively studied. A variety of protein and nonprotein toxins are found to modify the physiology of Na.sup.+ channels. At present, six different binding sites for toxins have been postulated. These include extracellular surface sites for tetrodotoxin and for two different classes of peptide toxins (.alpha.and .beta., usually isolated from scorpion venoms), intramembranous sites for three classes of lipophilic organic molecules (brevetoxin/ciguatoxin and the classical activators such as batrachotoxin and veratridine, and certain synthetic insecticides), and the site(s) of local anesthetics action. Each of these sites appear to be linked to at least one other site via conformationally coupled interactions that often are dependent on the membrane potential. Butterworth, J. T. and G. R. Strichartz, Anesthesiology, 72:711-734 (1990).
Many diseases of excitable tissues are known to be associated with, if not caused by, dysfunction of ion channels; these include cardiac arrhythmias, angina pectoris, cystic fibrosis, myotonia, and epilepsies, to mention only a few. A variety of drugs, for example, local anesthetics, antiarrhythmic agents, anticoconvulsants and psychoactive drugs, have been developed to treat these diseases. Channel
REFERENCES:
Butterworth IV, J.F. et al. "Mechanisms of Local Anesthesia: A Review". Anesthesiology 72(4):711-734 (1990).
Manavalan, P. et al. "Circular dichroism studies of acetylcholinesterase conformation. Comparison of the 11S and 5.6S species and the difference induced by inhibitory ligands". Biochimica Et Biophysica Acta 829(3):365-370 (1985).
Loret, E.P. et al. "An Anti-Insect Toxin Purified from the Scorpion Ancroctonus autralis Hector Also Acts on the .alpha.- and .beta.-Sites of the Mammalian Sodium Channel: Sequence and Circular Dichroism Study". Biochemistry 30:633-640 (1991).
Pennington, M.W. et al. "Synthesis and Biological Activiity of Six Monosubstituted Analogs of a Sea Anemone Polypeptide Neurotoxin". Peptide Research 3(5):228-231 (1990).
Wu, C.S et al. "Conformation of Acetylcholine Receptor in the Presence of Agonists and Antagonists". Journal of Protein Chemistry 9(1):119-126 (1990).
Darbon, H. et al. "Alpha-Scorpion neurotoxin derivatives suitable as potential markers of sodium channels". International Journal of Peptide and Protein Research 22(2):179-186 (1983).
Freschi, J.E. et al. "Effect of gamma radiation on sodium channels in different conformation in neuroblastoma cells". Biochimica Et Biophysica Acta 858(1):31-37 (1986).
Kitz, R.J. et al. "Conformational Changes of Acetylcholinesterase". Molecular Pharmacology 4(1):104-107 (1968).
Achutamurthy Ponnathapura
Haullin Albert P.
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