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

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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.