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

Abstract

Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.

α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

“…Solid-phase peptide synthesis was used to obtain α-conotoxins [V11L,V16D]ArIB [ 54 ], MII [ 98 ], PnIA [ 99 ] and RgIA [ 100 ]. α-Cobratoxin was isolated by the procedure described in [ 53 ] from Thailand cobra venom obtained from an authorized farm (Vinh Son, Vinh Tuong, Vinh Phuc province, Vietnam).…”

Mitochondrial Nicotinic Acetylcholine Receptors Support Liver Cells Viability After Partial Hepatectomy

“…Rabbit cyt c -specific antibodies were generated as previously described ( Gergalova et al, 2014 ). Conotoxin MII, synthesized according to Surin et al (2012) and α-conotoxin PeIA, synthesized as described ( Koval et al, 2011 ) was a kind gift of Prof. V. Tsetlin (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Russia). Dexalgin (analog of Dexketoprofen , (2 S )-2-[3-(benzoyl)phenyl]propanoic acid) was purchased from the public drugstore in Kiev.…”

Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors

“…When introducing bulk moieties into small peptides, a noticeable decrease in affinity, a change in specificity or an increase in non-specific interactions is more often observed, as we demonstrated earlier with the example of some photoactivated derivatives of α-conotoxins MI and GI ( Kasheverov et al, 2001 , 2006 ). However, fluorescent derivatives of some α-conotoxins were prepared, which became selective markers of different nAChR subtypes ( Hone et al, 2010 ; Surin et al, 2012 ; Barloscio et al, 2017 ). For instance, using a fluorescent derivative of α-conotoxin MII, a selective α6β2 nAChR antagonist, α6-containing nAChRs were localized in the retinal tissue and found to be predominantly localized to the ganglion cell layer ( Barloscio et al, 2017 ).…”