Studies on the structural basis for ion channel functions

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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.