Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-24
2003-11-04
Bugaisky, Gabrielle (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200, C436S501000
Reexamination Certificate
active
06641997
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a crystal of a cation channel protein, and methods of using such a crystal in screening potential drugs and therapeutic agents for use in treating conditions related to the function of such channels in vivo.
BACKGROUND OF INVENTION
Although numerous types of channel proteins are known, the main types of ion channel proteins are characterized by the method employed to open or close the channel protein to either permit or prevent specific ions from permeating the channel protein and crossing a lipid bilayer cellular membrane. One important type of channel protein is the voltage-gated channel protein, which is opened or closed (gated) in response to changes in electrical potential across the cell membrane. Another type of ion channel protein are celled mechanically gated channel proteins, for which a mechanical stress on the protein opens or closes the channel. Still another type is called a ligand-gated channel, which opens or closes depending on whether a particular ligand is bound the protein. The ligand can be either an extracellular moiety, such as a neurotransmitter, or an intracellular moiety, such as an ion or nucleotide.
Presently, over 100 types of ion channel proteins have been described, with additional ones being discovered. Basically, all ion channels have the same basic structure regarding the permeation of their specific ion, although different gating mechanisms (as described above) can be used. One of the most common types of channel proteins, found in the membrane of almost all animal cells, permits the specific permeation of potassium ions (K
+
) across a cell membrane. In particular, potassium ions permeate rapidly across cell membranes through K
+
channel proteins (up to 10
8
ions per second). Moreover, potassium channel proteins have the ability to distinguish among potassium ions, and other small alkali metal ions, such as Li
+
or Na
+
with great fidelity. In particular, potassium ions are at least ten thousand times more permanent than sodium ions. In light of the fact that both potassium and sodium ions are generally spherical in shape, with radii of about 1.33 Å and 0.95 Å respectively, such selectivity is remarkable.
Broadly, potassium channel proteins comprise four (usually identical) subunits. Presently two major types of subunits are known. One type of subunit contains six long hydrophobic segments (presumably membrane-spanning), while the other type contains two hydrophobic segments. Regardless of what type of subunits are used, potassium channel proteins are highly selective for potassium ions, as explained above.
Among their many functions, potassium channel proteins control the pace of the heart, regulate the secretion of hormones such as insulin into the blood stream, generate electrical impulses underlying information transfer in the nervous system, and control airway and vascular smooth muscle tone. Thus, potassium channels participate in cellular control processes that are abnormal, such as cardiac arrhythmia, diabetes mellitus, seizure disorder, asthma and hypertension, to name only a few.
Although potassium channel proteins are involved in such a wide variety of homeostatic functions, few drugs or therapeutic agents are available that act on potassium channel proteins to treat abnormal processes. A reason for a lack of presently available drugs that act on potassium channel proteins is that isolated potassium channel proteins are not available in great abundance, mainly because an animal cell requires only a very limited number of such channel proteins in order to function. Consequently, it has been very difficult to isolate and purify potassium channel proteins, reducing the amount of drug screening efforts in search of potassium channel protein acting drugs.
Hence, what is needed is accurate information regarding the structure of cation channel proteins so that drugs or therapeutic agents having an appropriate structure to potentially interact with a cation channel protein can be selected.
What is also needed is an ability to overcome the physical limitations regarding the isolation and purification of cation channel proteins, particularly potassium ion channel proteins.
What is also needed is a reliable method of utilizing cation channel proteins in screening potential drugs or agents for their possible use in treating conditions related to the function of cation channel proteins in vivo.
What is also needed are novel methods of using accurate information regarding the structure of cation channel proteins so that drugs or therapeutic agents can be screened for potential activity in treating abnormal control processes of the body.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
There is provided, in accordance with the present invention, a method of preparing a functional cation channel protein for use in an assay for screening potential drugs or other agents which interact with a cation channel protein, which permits the screening of potential drugs or agents that may be used as potential therapeutic agents in treating conditions related to the function of cation channel proteins in vivo.
More specifically, the method comprising the steps of providing a functional cation channel protein, conjugating the functional cation channel protein to a solid phase resin, contacting the potential drug or agent to the functional cation channel protein conjugated to the solid phase resin, removing the functional cation channel protein from the solid phase resin, and determining whether the potential drug or agent is bound to the cation channel protein.
In particular, the present invention extends to a method of preparing a functional cation channel protein for use in an assay as described above, wherein the providing step of the method comprises expressing an isolated nucleic acid molecule encoding the cation channel protein in a unicellular host, such that the cation channel protein is present in the cell membrane of the unicellular host, lysing the unicellular host in a solubilizing solution so that the cation channel protein is solubilized in the solution, and extracting the cation channel protein from the solubilizing solution with a detergent. In a preferred embodiment, the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:17, or degenerate variants thereof, or an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule having a DNA sequence of SEQ ID NO:17, or degenerate variants thereof.
Numerous methods of lysing a unicellular host are known to the skilled artisan, and have applications in the present invention. In a preferred embodiment, lysing the unicellular host in a solubilizing solution comprises sonicating the unicellular host in a protein solubilizing solution comprising 50 mM Tris buffer, 100 mM KCl, 10 mM MgSO
4
, 25 mg DNAse, 1, 250 mM sucrose, pepstatin, leupeptin, and PMSF, pH 7.5.
Furthermore, a skilled artisan is aware of numerous detergents that can be used to extract an integral membrane bound protein, such as a cation channel protein, from a solubilizing solution described above. Examples of such detergents include SDS, Triton-100, Tween 20, Tween 80, glycerol, or decylmaltoside, to name only a few. Preferably, 40 mM decylmaltoside is used to extract the cation channel protein from the solubilizing solution.
Moreover, numerous solid phase resins to which a functional cation channel protein can be conjugated have applications in a method of preparing a functional cation channel protein for use in an assay, as described above. For example, a solid phase resin comprising insoluble polystyrene beads, PVF, polyethylene glycol, or a cobalt resin, to namely only a few have application in the present invention. Preferably, a cation channel protein is conjugated to a cobalt resin at a protein to resin ratio that allows for saturation of t
Bugaisky Gabrielle
Klauber & Jackson
The Rockefeller University
LandOfFree
Assays for screening compounds which interact with cation... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Assays for screening compounds which interact with cation..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assays for screening compounds which interact with cation... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3138709