Probing the Toxin Binding Site of Sodium Channel with new Photoaffinity Labeling Reagents

本研究首次从cDNA克隆中推导出了钠离子通道蛋白的一级结构，并为其它离子通道蛋白提供了完整的序列信息。对这些离子通道的化学研究依赖于使用一些特定的神经毒素，这些神经毒素在受体位点作为通道结构和功能的分子探针。这些毒素的光反应衍生物是在分子水平上分析结合位点的潜在有用工具。新的光反应性基团（1）合成了一系列烷氧基苯基二氮杂环丙烯类光反应性卡宾前体。苯氧基二氮杂环丙烷的硝化，碘化，或钯催化的羰基化，得到了几个有用的光亲和标记的二氮杂环丙烷。还制备了这些二氮杂环丙烷的几种放射性标记的类似物。(2)合成了一系列氟代硝基苯氧基衍生物作为亲核选择性光反应基团用于光标记. Na^+通道的光亲和标记（1）合成了一种肽毒素μ-芋螺毒素GIIIA作为钠通道特异性配体。这种毒素的光反应性衍生物携带显色的二氮丙啶已被制备为光亲和标记试剂的肌肉型钠通道。试剂竞争性抑制石房蛤毒素与鳗鱼钠通道的结合。鳗鱼钠通道的光亲和标记是用<14>光反应性芋螺毒素的放射性14 C类似物进行的，放射性被特异性地掺入通道蛋白中。(2)河豚毒素的氟-硝基苯氧基衍生物的光标记的结果表明，有很少的亲核试剂在结合位点反应。

The primary structure structure of the sodium channel protein from eel electroplax was first deduced from cDNA clone, and complete sequence information is also available for other ion channels. Chemical studies of these ion channels are dependent upon use of a number of specific neurotoxins that act at the receptor sites as molecular probes of channel structure and function. Photoreactive derivatives of those toxins are potentially useful tools for structural analyses of the binding sites at the molecular level.1. New photoreactive groups (1) A series of alkoxyphenyldiazirines was synthesized as new photoreactive carbene precursors. The thallation of phenoxydiazirines followed by nitration, iodination, or palladium-catalyzed carbonyltion gave several useful diazirines for photoaffinity labeling. Several radiolabeled analogues of these diazirines were also prepared. (2) A series of fluoro-nitrophonoxy derivatives was synthesized as nucleophile selective photoreactive groups for photolabeling.2. Photoaffinity labelins of Na^+ channel (1) A peptide toxin, mu-conotoxin GIIIA, was synthesized as a sodium channel specific ligand. Photoreactive derivatives of this toxin carrying a chromogenic diazirine have been prepared as photoaffinity labeling reagents for muscle-type sodium channels. The reagents competitively inhibited te binding of saxitoxin to the eel sodium channel. Photoaffinity labeling of the eel sodium channel was performed with a radioactive ^<14>C analog of photoreactive conotoxin, and the radioactivites were specifically incorporated into the channel protein. (2) The results of photolabeling with fluoro-nitrophonoxy derivatives of tetrodotoxin suggest that there are very few nucleophiles to react at the binding site.