Studies on the tertiary structure and operational mechanism of ionic channels

我们在CHO细胞中表达了毒蕈碱型乙酰胆碱受体（mAChR I）和烟碱型乙酰胆碱受体亚单位，并对产物进行了纯化和表征。我们还尝试了杆状病毒系统，结果证明效率更高。使用纯化的样品，我们研究了结晶的蛋白质的条件，通过分析位点特异性突变的尼古丁乙酰胆碱受体，我们表明，在中间环的残基是一个决定因素的离子选择性，和不带电的极性残基的环状结构，连同相邻的中间环，形成一个短通道收缩接近细胞质侧的膜。我们分析了由牛肌肉α-、β-和δ-亚基组成的乙酰胆碱受体通道的自发开放。我们扩展了我们以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酶簇突变的影响。 ...更多信息 由于位于重复序列I-IV的SS 2区。这有力地支持了SS 2区域的每个重复形成的永恒的口和/或孔壁的channel.From骨骼和心脏二氢吡啶（DHP）receptors的一系列嵌合体结构的分析，我们证明了重复II和III之间的区域是至关重要的神经元型兴奋-收缩耦合，重复I确定嵌合受体的激活动力学。我们发现，通过表达DHP受体恢复膜内电荷运动的基因缺陷小鼠的肌管，表明DHP受体是密切相关的电压敏感机制。我们克隆了一个脑钙通道，并确定了它的一级结构。在非洲爪蟾卵母细胞中，骨骼肌α_2-和β-亚基的共表达极大地增强了该通道的功能表达。该通道是高阈值钙通道，对硝苯地平和ω-芋螺毒素不敏感。印迹分析表明它主要表达于小脑浦肯野细胞和颗粒细胞。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