Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Kyoto University|
IMOTO Keiji Kyoto University Faculty of Medicine, 医学部, 助教授 (00176512)
TSIEN Richard W. Stanford University, Department of Molecular and Cellular Physiology, 教授
BEAM Kurt G. Colorado State University, Department of physiology, 生理学教室, 教授
STUEHMER Wal マックスプランク生物物理化学研究所, 膜生物物理部門, 研究員
MICHEL Hartmut Max-Planck-Instituet for Biophysik, Abteilung Moleckulare Membrandiochemie, 分子膜生化学部門, 部長
MORI Yasuo Kyoto University Faculty of Medicine, 医学部, 助手 (80212265)
NAKAI Junichi Kyoto University Faculty of Medicine, 医学部, 助手 (80237198)
FUKUDA Kazuhiro Kyoto University Faculty of Medicine, 医学部, 助手 (90199224)
STUMER Walter Max-Planck-Institute for Biophysikqlische Chemie, Abteilung Membranbiophysik
|Project Period (FY)
Completed(Fiscal Year 1992)
|Budget Amount *help
¥4,100,000 (Direct Cost : ¥4,100,000)
Fiscal Year 1992 : ¥4,100,000 (Direct Cost : ¥4,100,000)
|Keywords||Ion channel / cDNA expression / cDNA cloning / Slte-directed mutagenesis / Nicotinic acetycholine receptor / Calcium channel / Sodium channel / Ryanodine receptor|
Bovine skeletal muscle nicotinic acetylcoline receptor alpha-subunit was expressed using baculovirus-infected insect cells. This alpha-subunit protein was 2-3% of total cellular protein. The protein could be solubilized and purifeid. Purified protein was shown to be glycosilated, and showed a high-affinity activity to alpha-bungarotoxin.
By analyzing ion permeation properties of site-specifically mutated rat brain sodium channel ll, amino acid residues that from part of the selectivity filter of the sodium channel were identified. Those acid residues are located in the SS2 regions. Substitution of glutamic acid residues for lysine and alanine in these critical regions conferred calcium channel properties on the sodium channel.
The channel pore size was estimated for site-specifically mutated nicotinic acetylcholine receptor channel. The results suggested that both of the pore size and the fixed charge are determinant of permeability, end that changes in charge may affect permeability by altering the pore size.
Two new types of barin calcium channel (Bll and Blll) were cloned. They are similar to the Bl calcium channel in amino acid seqence, and those three calcium channel from a subfamily which is distinct from dihydropyridine-sensitive L-type calcium. The Blll calcium was expressed using myotube from mice with muscular dysgenesis. The current was sensitive to omega-conotoxin, and its single-channel conductance was about 16 PS. These results indicated that Blll calcium channel is N-type.
A novel type of ryanodine receptor/calcium release channel was cloned, and its primary structure was determined. This receptor is strusturally similar to the skeletal muscle and cardiac conuterparts. The brain type ryanodine receptor is abundantly expressed in hippocampus, striate body, and thalamus, while the cardiac type of ryanodine receptor is more diffusely distributed in the brain. The brain type was expressed in tissues containing smooth muslce.