Synthesis of fluorescent analogs of alpha-conotoxin MII.
Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.
Vishwanath
Vijay A
VA
Interdepartmental Program in Neuroscience and Departments of Psychiatry and Biology, University of Utah, Salt Lake City, Utah 84112, USA.
McIntosh
J Michael
JM
D000818:Animals; D020916:Conotoxins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D015394:Molecular Structure; D018733:Nicotinic Antagonists / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D009865:Oocytes; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014982:Xenopus laevis
D020916:Conotoxins; D005456:Fluorescent Dyes; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C098872:alpha-conotoxin MII