The molecular mechanisms by which alcohols exert their actions remain poorly understood. Defining the target(s) is the primary step in elucidating the mechanism of alcohol action. Alcohols have been shown to alter the function of numerous types of voltage- and ligand-gated ion channels in the central nervous system and to affect multiple intracellular signaling pathways (Diamond and Gordon 1997; Harris 1999). However, no single target is well defined and fully characterized. It is believed that alcohol could act on multiple targets in the brain (Harris et al. 2008).
At relatively low concentrations, ethanol directly affects the function of many central and peripheral synapses (Liu and Hunt 1999). Post-synaptically, ethanol modulates the functions of a number of neurotransmitter receptors, such as GABA, glutamate, and serotonin (Diamond and Gordon 1997). Although clear effects of ethanol have been identified in post-synaptic compartments, recently there has been increasing evidence of a significant effect of ethanol on pre-synaptic function at concentrations well below 100 mM (Diamond and Gordon 1997). Evidence has emerged indicating that ethanol may be directly affecting synaptic transmission by altering vesicle fusion. A single amino acid polymorphism in the Munc18-SNARE complex associated protein correlates with strong differences in ethanol preference in a two-bottle choice assay (Fehr et al. 2005). When this analogous Unc18 mutation was created in the Caenorhabditis elegans, the resulting animals were resistant to both stimulatory and sedative effects of ethanol (Graham et al. 2009). This missense mutation in Unc18 also lengthened the duration of quantum release and slowed the frequency of release, indicating that ethanol may act by affecting synaptic vesicle exocytosis (Graham et al. 2009). However, the mechanism by which ethanol may affect synaptic release is unknown.
Munc13-1 is a pre-synaptic active-zone protein essential for synaptic vesicle fusion (Betz et al. 1997; Sassa et al. 1999) and neurotransmitter release, (Betz et al. 1998; Brose et al. 2000) and interact with both syntaxin and Munc18 proteins in mammalian brain. It is also implicated in modulating short-term pre-synaptic plasticity (Rosenmund et al. 2002).
Structurally, Munc13-1 is a large ~ 200 kDa peripheral membrane protein with multiple regulatory domains. These domains include an N-terminal Ca2+-binding C2 domain, a high-affinity diacylglycerol (DAG)/phorbol ester-binding C1 domain next to another C2 domain, two Munc13 homology domains, and a C-terminal C2 domain (Fig. 1) (Aravamudan et al. 1999). The binding of DAG or phorbol ester to the Munc13-1 C1 domain stimulates synaptic vesicle priming as well as the Munc13-1 translocation from the cytoplasm to the plasma membrane (Andrews-Zwilling et al. 2006). There are strong sequence similarities among the C1 domains of Munc13, Dunc-13, and Unc13 and the C1 domain of protein kinase C (PKC)s. The Munc13-1 and Dunc-13 C1 domains are particularly well conserved: 90% identical and 96% similar. Structurally, the C1 domain of the Munc13-1 proteins are similar to that of PKC, both having two β-sheets, a short C-terminal α-helix and two Zn+2-binding sites (Zhang et al. 1995; Shen et al. 2005). DAG binds inside a groove formed by two ‘rabbit-ear’ loops of Unc13, similar to the binding sites in PKCδ. The binding of DAG to the Unc13 C1 domain lowers the energy barrier for vesicle fusion and promotes neurotransmitter release (Basu et al. 2007). The H567K mutation at the beginning of the C1 domain prevents the binding of phorbol esters, thereby inhibiting the activation of vesicle fusion (Betz et al. 1998; Basu et al. 2007).
Figure 1. Munc13-1 domains and sequences. (a) Schematic representation of the domains of Munc13-1. The C1 regulatory domain binds to lipids, diacylglycerol, and phorbol esters; the C2 regulatory domain binds to anionic lipids and Ca2+; MHD1 and MHD2 represent Munc13 homology domains and suggested to bind syntaxin, the vesicle fusion protein. (b) Sequence comparison of different C1 domains. Residues that are conserved in each protein are highlighted in yellow. Residues that are conserved in all family members using the BLOSUM amino acid matrix criteria (Henikoff and Henikoff 1992) are highlighted in gray. The alcohol-binding residue found in this study is marked with a red colored box.
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PKCε knockout mouse consumes 75% less alcohol compared with the wild-type mouse (Hodge et al. 1999). We recently identified the PKCε C1 domain as a site of alcohol binding (Das et al. 2009). In PKCε, alcohol molecules bind to the His-248 and Tyr-250 residues, resulting in the inhibition of the PKCε activity, presumably by interfering with DAG binding and activation. The binding of alcohol with the PKCε C1 domain suggested that the Munc13-1 C1 domain may likewise be a target for alcohol binding. In addition, Unc13, the C. elegans homolog of Munc13-1, has been implicated for its sensitivity to volatile anesthetics (Metz et al. 2007). To explore if alcohol-binding site could be present in other proteins expressed in brain containing structurally related C1 domains, we chose to investigate the alcohol binding to the C1 domain of Munc13-1.
In this study, we expressed the C1 domain of Munc13-1 in E. coli, purified, and studied it's interaction with alcohols by fluorescence, photolabeling, and mass spectrometry and circular dichroism spectroscopy. Our results show that ethanol, butanol, and octanol interact with Munc13-1 C1 domain and the diazirine derivatives of butanol and octanol photoincorporate into a primary site consisting of Glu-16 in the C1 domain corresponding to the Glu-582 of the full- length protein. Mutation of this residue with alanine, leucine, or histidine affected the alcohol binding, indicating the role of glutamate residue in alcohol binding of Munc13-1 C1. We further test the hypothesis that Munc13-1 impacts the neural effects of alcohol using Drosophila melanogaster. Flies with reduced activity of the Dunc-13 ortholog display significantly increased preference for ethanol containing food compared with wild-type controls. This increased preference for ethanol is rescued by the expression of the rat Munc13-1 within the Drosophila nervous system. The functional complementation of the Dunc-13 alcohol self-administration phenotype by the vertebrate Munc13-1 indicates that this protein functions in vivo to modulate ethanol-related behaviors. Together, these data point to Munc13-1 as an impactful mediator of the pre-synaptic effects of ethanol.
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The major finding of this study is that pre-synaptic Munc13-1 binds alcohol at Glu-582 of its C1 domain and its partial knockdown alters alcohol self-administration in Drosophila.
Inspection of Glu-582 in the Munc13-1 C1 structure (Shen et al. 2005) reveals that it is located near an α-helix formed by residues 608CQDLL612 and on the tip of the ß-turn formed by residues 579YCYECE584 (Fig. 7). The hydrophilic Glu-582 is surrounded by hydrophobic residues such as Tyr-581, Tyr-583 of the ß-turn and Leu-611, Leu-612 of the α-helix, and hydrophilic residues such as Glu-584 of ß-turn and Gln-609, Asp-610 of α-helix indicating that the alcohol-binding site is amphiphilic in nature (Fig. 7). Some of these residues are located within the possible interaction radius (3Å) of Glu-582, raising the possibility of formation of a hydrogen bond network. The presence of the alcohol-binding residue in a turn or loop segment that is adjacent to an α-helix is one of the characteristic features of an alcohol-binding motif (Dwyer and Bradley 2000). The alcohol molecule could be involved in polar and non-polar interaction within an amphiphilic binding cavity, including the presence of weak hydrogen bond interaction with an amino acid and a water molecule (Dwyer and Bradley 2000). Detection of several photolabeled residues in the loop 579YCYECE584, other than Glu-582 (Table 2), is another indication for the existence of this alcohol-binding motif. In addition to this, our observation that the increase in melting temperature (monitored at 222 nm) in the presence of alcohols (Figure S6, Table S2) suggests that there may be a change in the α-helix conformational state and azialcohol labeling resulting in the multiple residues of the loop and the helix including Glu-582.
Figure 7. Putative alcohol-binding site in Munc13-1 C1. Location of Glu-582 in the C1 domain. (a) Ribbon diagram showing of the Munc13-1 C1 domain and alcohol-binding residue Glu-582 (red). α-helix important for alcohol recognition are depicted as a cylinder (split pea). Two zinc atoms that coordinated with cysteine and histidine residues are shown as spheres (gray). The ß-sheets, important structural element are shown in yellow color. (b) Surface diagram of Munc13-1 C1 showing the photolabeled residue Glu-582 (red). The models are generated using the solution structure of Munc13-1 C1 (Shen et al. 2005) and PyMol molecule visualization software.
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Glu-582 is not a conserved residue among the proteins containing C1 domains (Fig. 1b). While for PKCεC1B, the alcohol-binding site is composed of His-248 and Tyr-250, for PKCδC1B it is the Tyr-236, which is homologous to His-248 of PKCε. The 1.3Å crystal structure of PKCδC1B–cyclopropane methanol complex (Shanmugasundararaj et al. 2012) revealed that the alcohol-binding groove consists of Tyr-236, Met-239, and Ser-240. The hydroxyl group of cyclopropane methanol is hydrogen bonded to the nearby Tyr-236 and a water molecule with distances of 2.8Å and 3.0Å, respectively. Meanwhile, the methylene groups of cyclopropane ring interact (van der Waals interactions) with the methylene groups of Met-239 and Ser-240. Although Glu-582 in Munc13-1 and Tyr-236 in PKCδC1B may be involved in a similar type of hydrogen bond and similar hydrophobic interactions, a distinct difference is that, unlike in Munc13-1, where the zinc atom is only 3.3 Å away from the Glu-582, the closest distance between the zinc atom and Tyr-236 in PKCδC1B is about 13.8 Å, which rules out any possibility of zinc–alcohol interactions. The other difference is that—whereas Glu-582 in Munc13-1C1 is located far from the phorbol ester-binding loops—Tyr-236 in PKCδ is located close to it.
The involvement of glutamic acid in alcohol binding previously has been highlighted for several proteins, including Glu-262 for acetylcholine receptor (Pratt et al. 2000); Glu-33 for L1 cell adhesion molecule (Arevalo et al. 2008); Glu-163 and Glu-193 for Rho GDP dissociation inhibitor (Ho et al. 2008); Glu-146 for lignin peroxidase (Ambert-Balay et al. 1998); and Glu-13 for pepsin (Andreeva et al. 1984).
Recent studies indicate that Munc13-1 interacts with other proteins, such as Munc 18 and PKC directly or indirectly in regulating vesicle fusion and trafficking (Betz et al. 1997; Guan et al. 2008; Rizo and Rosenmund 2008). That PKC (Newton and Ron 2007; Qi et al. 2007) and Munc 18/Unc18 (Graham et al. 2009) are known to regulate alcohol actions and interact with Munc13-1 directly or indirectly, Munc13-1 can act as a key component in the protein milieu regulating the alcohol's action in the synapse. Furthermore, the close proximity of Glu-582 to the His-567 (8.8Å), which is known to affect the vesicle transfer and neurotransmitter release in pre-synaptic neurons (Betz et al. 1998), indicates that alcohol binding could also affect these processes.
A role for Munc13-1 as an effector of alcohol's pre-synaptic actions is supported by the Dunc-13 haploinsufficiency for ethanol self-administration. Dunc-13 has a conserved function in regulating pre-synaptic release and the C1 domain of Mun13-1 and Dunc-13 are strongly conserved (Aravamudan et al. 1999). Ethanol preference for Drosophila is thought to arise from the hedonistic properties of this drug (Devineni and Heberlein 2009, 2010; Xu et al. 2012). The functional complementation of the reduced Dunc-13 activity by the rat Munc13-1–EGFP fusion protein strongly suggests that the increased ethanol preference found in the Dunc-13P84200/+ heterozygotes is owing to the reduced Dunc-13 activity. Hence, Dunc-13 activity regulates ethanol self-administration. Interestingly, even though a reduction in Dunc-13 leads to an increase in ethanol preference, the reversal of this phenotype depends on the activity on Munc13-1, suggesting that Drosophila can be used as an in vivo model for measuring Munc13-1 activity and the impact of ethanol binding to the C1 domain.