Synthesis of Fluorescent Analogs of α-Conotoxin MII

Abstract

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.

Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation

“…The fraction eluted with 100% methanol was reduced under vacuum, resuspended in water and chromatographed on a C18 column using a Gilson HPLC system, being finally eluted with a 50 m m sodium phosphate buffer (pH 6.5)/methanol gradient. Peptide concentrations were determined by measurement of the absorbance at 278 nm [32].…”

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

“…Fluorescently-labeled analogs of α-CTx MII have also recently been synthesized (78). Using the radiolabeled α-CTx MII, Quik and co-workers have found a selective downregulation of α-CTx MII binding sites within rodent and monkey striatum after nigorstriatal damage (79–82) as well as in humans with Parkinson’s (82).…”

The effect of MII α-conotoxin and its N-terminal derivatives on Ca2+- and Na+-signals induced by nicotine in SH-SY5Y neuroblastoma cells

“…For example, substitution of an alanine residue for a glycine residue caused a 4.5 fold increase of the IC 50 value [22], while the addition of fluorescent labels with a mass amounting to 10-15% of the actual toxin mass to the terminal amino group caused a 40-60 fold increase in IC 50 [28]. We synthe sized analogues of MII α conotoxin with the N termi nal glycine residue labeled with fluorescein isothiocyan ate (FITC MII) or Bolton Hunter reagent (MII BH).…”

“…A radioactively labeled derivative of MII α conotoxin analogue containing an additional tyrosine residue at the N terminus (Y 0 MII) was used for the quantification of the α6 subunit in mouse brain preparations [25][26][27]. Synthesis of a range of MII α conotoxin derivatives bearing various fluorescent labels has been reported [28], but the data concerning the use of these deriva tives for studying natural objects is missing.…”