Mechanism of action of presynaptic toxins from the coral snake Micrurus lemniscatus lemniscatus venom on acetylcholine release and ion channel modulation

• Grant number: 16/23432-1
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: March 01, 2017
• End date: February 21, 2018
• Field of knowledge: Biological Sciences - Pharmacology - Toxicology
• Principal Investigator: Stephen Hyslop
• Grantee: Rafael Stuani Floriano
• Supervisor: Edward Gerard Rowan
• Host Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: University of Strathclyde, Scotland
• Associated to the scholarship: 14/24409-8 - Characterization of presynaptic toxins from venom of the coral snake Micrurus lemniscatus lemniscatus, BP.PD

Abstract

Coral snake (Micrurus spp.) venoms are known primarily for their neurotoxicity, which is mediated primarily by alpha-neurotoxins with postsynaptic activity and beta-neurotoxins (neurotoxic phospholipases - PLA2) with presynaptic activity. Preliminary studies have shown that venom of the coral snake Micrurus lemniscatus lemniscatus exerts presynaptic activity involving at least two PLA2 neurotoxins. In this work, we intend to purify and biochemically characterize these toxins using a variety of electrophysiological approaches (measurement of membrane and endplate potentials, extracellular recordings of compound action potential and perineural waves, and intracellular recordings using patch-clamp) and cellular assays (mobilization of intracellular Ca2+ and immunohistochemical analyses) in tissues exposed to the toxins. Specifically, with regard to the period to be spent abroad in this BEPE, the major aim is to receive training and gain experience in the design and execution of patch-clamp experiments. This will be achieved by investigating the presynaptic activity of M. l. lemniscatus toxins on various aspects of peripheral neurotransmission. Specifically, the following aspects will be investigated: (1) cultured DRG cells will be used to examine the activity of the toxins on isolated channel currents in a whole-cell patch-clamp configuration, (2) DRG cells and mouse triangularis sterni nerve-muscle preparations will be used to assess the role of intracellular Ca2+ mobilization in response to the toxins by using fluorescent Ca2+ imaging, and (3) immunohistochemical analysis will be used to examine the expression of synaptic vesicle and pre- and post-synaptic proteins involved in neurotransmitter release (exocytosis) in mouse phrenic nerve-diaphragm preparations pretreated with toxins; this analysis will also serve to assess the morphological intactness of the motor endplate region. The results obtained in this study should contribute to our understanding of the mechanisms involved in the presynaptic activity of M. l. lemniscatus venom. (AU)