Biotinylated derivatives of omega-conotoxins GVIA and MVIID: probes for neuronal calcium channels.
The omega-conotoxins are small, disulfide-rich peptides which inhibit voltage-sensitive calcium channels. Biotinylated omega-conotoxins are potentially useful reagents for characterizing distinct subsets of calcium channels. We describe the preparation and characterization of biotinylated derivatives of two specific omega-conotoxins, GVIA and MVIID, which bind different calcium channel subtypes. Eight biotinylated derivatives were tested; all specifically displaced binding of the radiolabeled unbiotinylated omega-conotoxin. In general, the addition of one biotin moiety decreased the apparent affinity for the receptor target site by only approximately 10-fold. However, derivatization of omega-conotoxin MVIID at the Lys10 residue caused a much more marked effect, a ca 500-fold decrease in affinity. These results indicate that the vicinity of the Lys10 residue of omega-conotoxin MVIID may be more critical for binding to the receptor target site than regions around other amino groups in omega-conotoxins GVIA and MVIID. Thus, high affinity biotinylated omega-conotoxin GVIA and MVIID derivatives have been chemically defined; the biotin groups have been shown to be accessible to streptavidin. Given the commercial availability of streptavidin coupled to various reporter groups, the biotinylated omega-conotoxin derivatives described here should be widely useful for fluorescence, electron microscopic or immunological applications.
Haack
J A
JA
Biology Department, University of Utah, Salt Lake City 84112.
Kinser
P
P
Yoshikami
D
D
Olivera
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001522:Behavior, Animal / Q000187:drug effects; D001710:Biotin / Q000737:chemistry; D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins; D004594:Electrophysiology; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D019809:Streptavidin; D013569:Synapses / Q000187:drug effects; D020866:omega-Conotoxin GVIA
D001426:Bacterial Proteins; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020916:Conotoxins; D010455:Peptides; D001360:Avidin; C085882:omega-conotoxin MVIID; D001710:Biotin; D019809:Streptavidin; D020866:omega-Conotoxin GVIA