Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-07-21
2001-12-04
Stucker, Jeffrey (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070300, C435S320100, C536S023500
Reexamination Certificate
active
06326168
ABSTRACT:
TECHNICAL FIELD
The present invention belongs to the field of genetic engineering, and particularly relates to a novel potassium channel protein expressed exclusively in the brain, or an equivalent thereof, a gene encoding the protein or an equivalent thereof, a vector containing the gene, a host cell containing the vector, and so on.
BACKGROUND ART
Potassium channel is a protein which is distributed in the surface membrane of cells and selectively allows potassium ions to pass through it, and it is considered that it takes an important role in controlling membrane potential of cells. Particularly, in nerve and muscle cells, it contributes to the neurotransmission of central and peripheral nerves, pace making of the heart, contraction of muscles and the like by controlling frequency, persistency and the like of action potential. In addition, it has been shown that it is also concerned in the secretion of hormones, adjustment of cell volume, proliferation of cells and the like.
As the classification based on the opening and closing mechanism of the channel, a voltage-dependent potassium channel, an inwardly rectifying potassium channel, a calcium-dependent potassium channel, a receptor coupling type potassium channel and the like have so far been identified. In addition, an ATP-dependent potassium channel, a pH-dependent potassium channel and the like have also been reported. Among them, the voltage-dependent potassium channel has a characteristic in that it opens when the membrane potential is depolarized. In general, potassium ions are present in a non-equilibrium state of about 5 mM outside the cell and about 150 mM inside the cell. Thus, when the voltage-dependent potassium channel is opened by depolarization, potassium ions flow out from intracellular part to extracellular part and, as a result, induce restoration of the membrane potential (re-polarization). Accordingly, the opening of voltage-dependent potassium channel induces reduction of excitability of nerve and muscle cells and the like. Also, it causes changes in cellular functions in non-excitatory cells too, such as increase in the driving force for Ca
2+
and subsequent increase in the flow of the same ion into the intracellular part. A compound capable of modifying opening of the voltage-dependent channel has a possibility of controlling various functions of cells, including excitability of nerve and muscle cells.
Genes of some types of the voltage-dependent potassium channel have been isolated from the brain and heart, and primary structure of the protein has been revealed. Based on the primary structure, it has been suggested that the voltage-dependent potassium channel has six transmembrane domains (S1 to S6) and one ion permeation region (H5). Also, it is assumed that the fourth transmembrane domain S4 contains basic amino acids having positive charge at intervals of 3 to 4 bases and functions as a voltage sensor.
These channels are roughly divided into Shaker type and eag type, based on the similarity of amino acid sequences. The Shaker type is a family having markedly high diversity and can be further divided into four groups of Kv1, Kv2, Kv3 and Kv4. On the other hand, the eag type is constituted by eag, eag-related gene and elk, and it related genes include hyperpolarization activation type potassium channels corresponding to KAT gene cluster and a cation channel which is activated by a cyclic nucleotide.
Regarding the importance of voltage-dependent potassium current in the brain, several findings have been obtained using these cloned voltage-dependent potassium channels. For example, relationship of Kv1.1 with memory and pain has been suggested by antisense-aided in vivo experiments (Meiri, N. et al. (1997)
Proc. Natl. Acad. Sci. USA
, 94, 4430-4434; Galeotti, N. et al. (1997)
J. Pharmaco. Exp. Ther
., 281, 941-949). Regarding Kv3.1, its participation in the excitability of GABA activating interneuron in cerebral cortex has been shown (Massengill, J. et al. (1997)
J. Neurosci
., 3136-3147). On the other hand, some experiments carried out using tetraethylammonium and 4-aminopyridine as non-selective inhibitors of the voltage-dependent potassium channel have also been reported. It has been shown that tetraethylammonium suppresses voltage-dependent potassium current in cerebral cortex nerve cells and also inhibits apoptosis of the same nerve cells (Yu, S. P. et al. (1997),
Science
, 278, 114-117). Also, it is known that intraventricular administration of 4-aminopyridine causes epileptic attack. These results suggest a possibility that an agent capable of controlling the activity of voltage-dependent potassium channel in the brain will become a therapeutic agent for central nervous system disorders such as dementia due to disturbance of memory and so on, nerve cell death accompanied by cerebral ischemia, epilepsy and the like.
On the other hand, most of the voltage-dependent potassium channels so far cloned are distributed in a large number of tissues among organs in the whole body. Thus, even when an agent which acts selectively on a specified voltage-dependent potassium channel is found, there is a possibility that the agent acts on many tissues and thereby induces originally unexpected agent effects. In order to find an agent having less side effects by targeting a potassium channel, it is necessary to clone a potassium channel in which its expressing tissue is restricted.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a novel potassium channel protein expressed exclusively in the brain, as a target of therapeutic agents for central nervous system disorders such as dementia, cerebral ischemic disorder, epilepsy and the like, and another object of the present invention is to provide a method for screening compounds and peptides capable of modifying activity of the same potassium channel protein, which are useful as therapeutic agents for central nervous system disorders, and a novel agent for use in the treatment of central nervous system disorders, which specifically acts upon the central nervous system and generates less side effects.
With the aim of solving the aforementioned problems, the present inventors have conducted intensive studies and, as a result, succeeded in isolating a gene coding for a novel potassium channel protein expressed exclusively in the brain. The inventors have also succeeded in expressing the novel potassium channel protein expressed exclusively in the brain and establishing a method for the screening of compounds and peptides capable of modifying activity of the same potassium channel protein.
The present invention relates to:
1) a potassium channel protein or an equivalent thereof, which has an amino acid sequence selected from either of Sequence Nos. 2 and 6, or an amino acid sequence resulting from said amino acid sequence by substitution, deletion or insertion of certain amino acid(s), and is expressed exclusively in the brain,
2) the potassium channel protein or an equivalent thereof according to the item 1), which is expressed exclusively in the human brain,
3) a potassium channel protein which has an amino acid sequence selected from either of Sequence Nos. 2 and 6, 4) a gene which has a gene sequence encoding the potassium channel protein or an equivalent thereof described in the items 1) to 3),
5) a gene which has a gene sequence encoding an amino acid sequence selected from either of Sequence Nos. 2 and 6, 6) a gene which has a gene sequence selected from either of the 6th to 3257th gene sequence of Sequence No. 1 or the 4th to 3057th gene sequence of Sequence No. 5, or a gene which is degenerate with respect to said gene,
7) a vector which contains the gene of the items 4) to 6),
8) a host cell which contains the vector of the item 7), or
9) a method for producing the potassium channel protein described in the items 1) to 3), which uses the host cell of the item 8).
The terms to be used in the present invention are explained in the following. The term “substitution, deletion or insertion of amino acid” means that one or a plur
Miyake Akira
Mochizuki Shinobu
Yokoi Hiromichi
Seharaseyon Jegatheesan
Stucker Jeffrey
Sughrue Mion Zinn Macpeak & Seas, PLLC
Yamanouchi Pahrmaceutical Co., Ltd.
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