Studies on the tertiary structure and operational mechanism of ionic channels

我们在CHO细胞中表达了M胆碱型乙酰胆碱受体(MAChR I)和烟碱型乙酰胆碱受体亚基，并对产物进行了纯化和鉴定。我们还尝试了巴库洛病毒系统，结果证明它更有效。利用纯化的样品，我们研究了蛋白质结晶的条件。通过对定点突变的尼古丁乙酰胆碱受体的分析，我们发现中间环上的残基是离子选择性的决定因素，并且不带电荷的极性残基的环状结构与相邻的中间环一起，在膜的细胞质一侧附近形成了一个短通道收缩。我们分析了由牛肌肉α、β和β亚基组成的乙酰胆碱受体通道的自发开放。我们扩展了我们先前关于河豚毒素敏感性的工作，发现河豚毒素敏感性和单通道电导受到两个氨基酸簇resi…突变的影响位于重复序列I-IV的SS2区域的会费较多。这有力地支持了每个重复序列的SS2区域形成了通道的永恒嘴巴和/或孔壁。通过对一系列由骨骼和心脏二氢吡啶(DHP)受体组成的嵌合结构的分析，我们证明了重复序列II和III之间的区域对于骨骼类型的兴奋-收缩偶联是关键的，而重复序列I决定了嵌合受体的激活动力学。我们的研究表明，在发育不良的小鼠的肌管中，DHP受体的表达可以恢复膜内电荷的运动，这表明DHP受体与电压敏感机制密切相关。我们克隆了一个大脑钙通道，并测定了它的一级结构。该通道在非洲爪哇卵母细胞中的功能表达被骨骼肌α_2和β亚基共表达极大地增强。该通道是一种高阈值钙通道，对硝苯地平和欧米茄毒素不敏感。印迹分析表明，它主要在小脑浦肯野和颗粒细胞中表达。较少

We expressed muscarinic acetylcholine receptor (mAChR I) and nicotinic acetylcholine receptor subunits in CHO cells, and purified and characterized the products. We also tried the baculo virus system, which turned out to be more efficient. Using purified samples, we investigated conditions for crystallisations of the proteins.By analysing site-specifically mutated nicotine acetylcholine receptor, we showed that the residues in the intermediate ring is a determinant of ion selectivity, and that a ring-like structure of uncharged polar residues, together with the neighbouring intermediate ring, forms a short channel constriction close to the cytoplasmic side of the membrane. We analysed spontaneous openings of an acetylcholine receptor channel composed of bovine muscle alpha-, beta- and delta-subunits.We extended our previous work on tetrodotoxin sensitivity and found that tetrodotoxin sensitivity and single-channel conductance are affected by mutations in two clusters of amino acid resi … More dues locating in the SS2 region of repeats I-IV. This strongly supports that the SS2 region of each repeat forms the eternal mouth and/or the pore wall of the channel.From analysis of a series of chimeric constructs made of skeletal and cardiac dihydropyridine (DHP) receptors, we demonstrated that the region between repeats II and III is critical for skeletal-type excitation-contraction coupling, and that repeat I determines kinetics of activation of the chimeric receptors. We showed that intramembranous charge movement is restored by expressing DHP receptor in myotubes of dysgenic mice, indicating that the DHP receptor is closely related to the voltage-sensing mechanism. We cloned a brain calcium channel and determined its primary structure. Functional expression of this channel in Xenopus oocytes is enormously enhanced by coexpression of skeletal muscle alpha_2- and beta-subunits. This channel is a high-threshold calcium channel, insensitive to nifedipine and omega-conotoxin. Blot analysis suggests it is predominantly expressed in cerebellar purkinje and granular cells. Less