Studies on the tertiary structure and operational mechanism of ionic channels

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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