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乙酰碱受体α亚基。这种α -亚基蛋白占细胞总蛋白的2-3%。该蛋白可溶解纯化。纯化后的蛋白被证明是糖硅化的，并显示出对α -虫毒的高亲和力活性。通过分析位点特异性突变的大鼠脑钠通道ⅱ的离子渗透特性，鉴定了部分钠通道选择性过滤器的氨基酸残基。这些酸残基位于SS2区域。谷氨酸残基在这些关键区域取代赖氨酸和丙氨酸，赋予钠通道钙通道性质。对位点特异性突变的烟碱乙酰胆碱受体通道的孔径进行了估计。结果表明，孔隙大小和固定电荷都是渗透率的决定因素，电荷的变化可能通过改变孔隙大小来影响渗透率。克隆了两种新的钡钙通道（Bll和Bll）。它们在氨基酸序列上与Bl钙通道相似，并且这三个钙通道属于一个亚家族，与二氢吡啶敏感的l型钙不同。用肌发育不良小鼠的肌管表达Blll钙。该电流对ω - concontoxin敏感，其单通道电导约为16 PS，说明bll钙通道为n型。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。这种受体在结构上与骨骼肌和心脏器官相似。脑型ryanodine受体在海马、纹状体和丘脑中大量表达，而心型ryanodine受体在脑内的分布更为弥漫性。脑型在含有平滑肌的组织中表达。

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.