UNC-18 Modulates Ethanol Sensitivity inCaenorhabditis elegans

Abstract

Acute ethanol exposure affects the nervous system as a stimulant at low concentrations and as a depressant at higher concentrations, eventually resulting in motor dysfunction and uncoordination. A recent genetic study of two mouse strains with varying ethanol preference indicated a correlation with a polymorphism (D216N) in the synaptic protein Munc18-1. Munc18-1 functions in exocytosis via a number of discrete interactions with the soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) protein syntaxin-1. We report that the mutation affects binding to syntaxin but not through either a closed conformation mode of interaction or through binding to the syntaxin N terminus. The D216N mutant instead has a specific impairment in binding the assembled SNARE complex. Furthermore, the mutation broadens the duration of single exocytotic events. Expression of the orthologous mutation (D214N) in the Caenorhabditis elegans UNC-18 null background generated transgenic rescues with phenotypically similar locomotion to worms rescued with the wild-type protein. Strikingly, D214N worms were strongly resistant to both stimulatory and sedative effects of acute ethanol. Analysis of an alternative Munc18-1 mutation (I133V) supported the link between reduced SNARE complex binding and ethanol resistance. We conclude that ethanol acts, at least partially, at the level of vesicle fusion and that its acute effects are ameliorated by point mutations in UNC-18. INTRODUCTIONSynaptic transmission initially involves the fusion of vesicular and plasma membranes within the presynaptic terminal. Membrane-membrane fusion is driven by the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is composed at the mammalian synapse of syntaxin-1, vesicle-associated membrane protein (VAMP)-2 and synaptosome-associated protein of 25 kD Jahn and Scheller, 2006). Although SNARE proteins are regarded as the minimal machinery required for fusion (Weber et al., 1998), there exists a large degree of overlaying regulation by an abundance of interacting proteins. One such protein is Munc18-1, the mammalian homologue of the nSec-1/Munc18-1 (SM) protein family.Originally isolated via genetic screens in Caenorhabditis elegans and yeast (Brenner, 1974;Novick and Schekman, 1979), SM proteins are essential exocytotic proteins primarily characterized via their strong interaction with syntaxin. The conserved function for SM proteins remains controversial and includes syntaxin trafficking Arunachalam et al., 2008), vesicle recruitment and/or docking (Voets et al., 2001;Weimer et al., 2003), and the fusion process itself (Barclay et al., 2003;Shen et al., 2007). A consensus on SM protein function has been hampered in part due to a lack of consistency in the mechanism of its syntaxin interaction. Munc18-1 was originally described to bind syntaxin only when syntaxin adopted a closed conformation (referred to here as mode 1 binding) (Dulubova et al., 1999;Yang et al., 2000), consequently precludi...

It takes nerves to fight infections: insights on neuro-immune interactions fromC. elegans

“…Among its many effects, alcohol influences immune function mainly through the HPA: acute alcohol exposure stimulates the HPA to release neural immunomodulators (Haddad, 2004). Studies in C. elegans have identified presynaptic proteins, including RAB-3 and UNC-18, as ethanol targets, suggesting that ethanol influences neuronal function by affecting crucial steps of vesicle priming (Kapfhamer et al, 2008;Graham et al, 2009). The mouse RAB-3 ortholog also functions in ethanol sensitivity (Kapfhamer et al, 2008), suggesting a conserved mechanism by which ethanol affects neuronal function.…”

Nematodes feel a craving - Using Caenorhabditis elegans as a model to study alcohol addiction

“…Analysis of an alternative Munc18-1 mutation (I133V) supports the link between reduced SNARE complex binding and ethanol resistance [20] . ethanol as a result of prolonged pre-exposure to the drug [24] .…”

“…Downstream of BK and Ca 2+ channel function, ethanol may act on additional presynaptic effectors, such as Rab3 and Munc18, which function in vesicle fusion and recruitment/docking respectively, to modulate neurotransmitter release [18,20,29,30] ( Fig. 1).…”

A Novel Cholinergic Action of Alcohol and the Development of Tolerance to That Effect inCaenorhabditis elegans

“…Mutations in multiple genes have previously been shown to generate altered phenotypes compared to wild-type animals upon treatment with ethanol (Davies et al 2003(Davies et al , 2004Kayser et al 2003;Kapfhamer et al 2008;Graham et al 2009;Speca et al 2010;Bettinger et al 2012;Bhandari et al 2012;Jee et al 2013;Raabe et al 2014). We examined two of these genes that have robust mutant ethanol response phenotypes, slo-1 and npr-1.…”

“…C. elegans are sensitive to acute ethanol exposure, demonstrating an increased speed of locomotion at low ethanol concentrations (Graham et al 2009) and decreased amplitude of body bends and decreased rates of crawling, swimming, egg laying, and pharyngeal pumping at higher concentrations of ethanol (Davies et al 2003;Mitchell et al 2007;Speca et al 2010). Ethanol also produces disinhibition of certain crawling behaviors that would normally be inhibited in a liquid environment (Topper et al 2014).…”