Studies on the structural basis for ion channel functions

利用杆状病毒感染的昆虫细胞表达牛骨骼肌烟碱型乙酰胆碱受体α亚基。该α亚基蛋白占细胞总蛋白的2-3%。该蛋白具有良好的溶解性和纯净性。纯化的蛋白是糖化的，对α-银环蛇毒素具有高亲和力。通过分析定点突变的大鼠脑钠通道11的离子渗透特性，鉴定了来自钠通道部分选择性过滤器的氨基酸残基。这些酸残基位于SS2区。在这些关键区域用谷氨酸残基取代赖氨酸和丙氨酸赋予钠通道钙通道特性。对定点突变的烟碱型乙酰胆碱受体通道的通道孔径进行了估算。结果表明，孔大小和固定电荷都是决定渗透性的因素，电荷的变化可能通过改变孔大小来影响渗透性。它们在氨基酸序列上与Bl钙通道相似，而这三个钙通道属于一个亚家族，不同于二氢吡啶敏感的L钙。用肌管表达肌肉发育不全小鼠的BLL1钙。该电流对欧米茄毒素敏感，其单通道电导约为16ps。克隆了一种新型的Ryanodine受体/钙释放通道，并确定了其一级结构。这种受体在结构上类似于骨骼肌和心脏。脑型Ryanodine受体在海马体、纹状体和丘脑表达丰富，而心脏型Ryanodine受体在脑内分布较广。脑型在含有光滑肌肉的组织中表达。

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.