Keizer David W, West Peter J, Lee Erinna F, Yoshikami Doju, Olivera Baldomero M, Bulaj Grzegorz, Norton Raymond S
The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
J Biol Chem. 2003 Nov 21;278(47):46805-13. doi: 10.1074/jbc.M309222200. Epub 2003 Sep 10.
SmIIIA is a new micro-conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other micro-conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. Consistent with the absence of hydroxyprolines, SmIIIA adopts a single conformation with all peptide bonds in the trans configuration. The spatial orientations of several conserved Arg and Lys side chains, including Arg14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other micro-conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in micro-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other micro-conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp15 side chain, which is unique to SmIIIA. Arg17, which replaces Lys in the other micro-conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N- and C-terminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.
SmIIIA是最近从食粪芋螺中分离出的一种新型微芋螺毒素。尽管它与其他微芋螺毒素具有一些生化特性（半胱氨酸残基的排列以及一个被认为与通道孔附近残基相互作用的保守精氨酸），但它有几个独特的特征，包括不含羟脯氨酸，并且是第一个被鉴定的抗河豚毒素电压门控钠通道的特异性拮抗剂。因此，它是研究这些通道功能作用的一个潜在有用工具。我们已经确定了SmIIIA在水溶液中的三维结构。与不含羟脯氨酸一致，SmIIIA采用单一构象，所有肽键均处于反式构型。几个保守的精氨酸和赖氨酸侧链的空间取向，包括在钠通道结合中起关键作用的Arg14（使用一致编号系统），与其他微芋螺毒素中的相似，但N端区域不同，这反映了SmIIIA中第8位残基之前肽键的反式构象，而微芋螺毒素GIIIA和GIIIB中的是顺式构象。SmIIIA与其他微芋螺毒素表面的比较表明，SmIIIA对TTX抗性通道的亲和力受SmIIIA特有的Trp15侧链影响。在其他微芋螺毒素中取代赖氨酸的Arg17可能也很重要。与从结构得出的这些推论一致，对SmIIIA和PIIIA的两个嵌合体进行的实验，其中它们的N端和C端一半被重组，结果表明SmIIIA C端一半中的残基赋予了对青蛙交感神经元细胞体中抗河豚毒素钠通道的亲和力。SmIIIA和具有SmIIIA C端一半的嵌合体也抑制交感神经元节后轴突中的抗河豚毒素钠通道，这通过它们对在河豚毒素存在下仍持续的C神经元复合动作电位的抑制来表明。
