allows for reuptake of synaptobrevin for further use in vesicles, whereas the other snare proteins remain associated with the cell membrane. the dissociated snare proteins have a higher energy state than the more stable cis-snare complex. it is believed that the energy that drives fusion is derived from the transition to a lower energy cis-snare complex. the atp hydrolysis-coupled dissociation of snare complexes is an energy investment that can be compared to "cocking the gun" so that, once vesicle fusion is triggered, the process takes place spontaneously and at optimum velocity. a comparable process takes place in muscles, in which the myosin heads must first hydrolyze atp in order to adapt the necessary conformation for interaction with actin and the subsequent power stroke to occur. regulatory effects on exocytosis regulation via snap-25 palmitoylation the q-snare protein synaptosomal-associated protein 25 (snap-25) is composed of two α-helical domains connected by a random coil linker. the random coil linker region is most notable for its four cysteine residues. the α-helical domains combine with those of both syntaxin and synaptobrevin (also known as vesicle associated membrane protein or vamp) to form the 4-α-helix coiled-coil snare complex critical to efficient exocytosis. while syntaxin and synaptobrevin both contain transmembrane domains which allow for docking with target and vesicle membranes respectively, snap-25 relies on the palmitoylation of cysteine residues found in its random coil region for docking to the target membrane. some studies have suggested that association with syntaxin via snare interactions precludes the need for such docking mechanisms. syntaxin knockdown studies however, failed to show a decrease in membrane bound snap-25 suggesting alternate docking means exist. the covalent bonding of fatty acid chains to snap-25 via thioester linkages with one or more cysteine residues therefore, provides for regulation of docking and ultimately snare mediated exocytosis. this process is mediated by a specialized enzyme called dhhc palmitoyl transferase. the cysteine rich domain of snap-25 has also been shown to weakly associate with the plasma membrane possibly allowing it to be localized near the enzyme for subsequent palmitoylation. the reverse of this process is carried out by another enzyme called palmitoyl protein thioesterase (see figure). a simplified depiction of the palmitoylation of a cysteine residue in a protein the availability of snap-25 in the snare complex is also theorized to possibly be spatially regulated via localization of lipid microdomains in the target membrane. palmitoylated cysteine residues could be localized to the desired target membrane region via a favorable lipid environment (possibly cholesterol rich) complementary to the fatty acid chains bonded to the cysteine residues of snap-25. snap-25 regulation of voltage-gated ca2+ channels in neuronal axon terminals as an action potential reaches the axon terminal, depolarization events stimulate the opening of voltage-gated calcium channels (vgccs) allowing the rapid influx of calcium down its electrochemical gradient. calcium goes on to stimulate exocytosis via binding with synaptotagmin 1. snap-25 however, has been shown to negatively regulate vgcc function in glutamatergic neuronal cells. snap-25 leads to a reduction of current density through vgcc's and therefore a decrease in the amount of calcium that is binding the synaptotagmin, causing a decrease in neuronal glutamatergic exocytosis. conversely, underexpression of snap-25 allows for an increase in vgcc current density and increase in exocytosis. further investigation has suggested possible relationships between snap-25 over/underexpression and a variety of brain diseases. in attention-deficit/hyperactivity disorder or adhd, polymorphisms at the snap-25 gene locus in humans have been linked to the disease suggesting a potential role in its manifestation. this is further suggested by heterogeneous snap-25 knockout studies performed on coloboma mutant mice, which led to phenotypic characteristics of adhd. studies have also shown a correlation of snap-25 over/underexpression and the onset of schizophrenia. syntaxin and the habc domain syntaxin consists of a transmembrane domain (tmd), alpha-helical snare domain, a short linker region, and the habc domain which consists of three alpha-helical regions. the snare domain in syntaxin serves as a target site for docking of snap-25 and synaptobrevin in order to form the four helix bundle requisite to the snare complex and subsequent fusion. the habc domain, however, serves as an autoinhibitory domain in syntaxin. it has been shown to fold over and associate with the snare domain of syntaxin inducing a "closed" state, creating a physical barrier to the formation of the snare motif. conversely, the habc domain can again disassociate with the snare domain leaving syntaxin free to associate with both snap-25 and synaptobrevin. syntaxin 1b and readily releasable pool of vesicles there is an immense diversity of syntaxin subtypes, with 15 varieties in the human genome. it has been suggested that syntaxin1b has a role in regulating number of synaptic vesicles ready for exocytosis in the axon terminal. this is also called the readily releasable pool (rrp) of vesicles. a knock out study in 2014 showed that the lack of syntaxin1b led to a significant decrease in rrp size. toxins many neurotoxins directly affect snare complexes. such toxins as the botulinum and tetanus toxins work by targeting the snare components. these toxins prevent proper vesicle recycling and result in poor muscle control, spasms, paralysis, and even death. botulinum neurotoxin botulinum toxin (bont) is one of the most potent toxins to have ever been discovered. it is a proteolytic enzyme that cleaves snare proteins in neurons. its protein structure is composed of two peptide subunits, a heavy chain (100kdas) and a light chain (50kdas), which are held together by a disulfide bond. the action of bont follows a 4-step mechanism including binding to the neuronal membrane, endocytosis, membrane translocation, and proteolysis of snare proteins. target snare proteins of botulinum neurotoxin (bont) and tetanus neurotoxin (tent) inside the axon terminal. in its mechanism of action, the heavy chain of bont is first used to find its neuronal targets and bind to the gangliosides and membrane proteins of presynaptic neurons. next, the toxin is then endocytosed into the cell membrane. the heavy chain undergoes a conformational change important for translocating the light chain into the cytosol of the neuron. finally, after the light chain of bont is brought into the cytosol of the targeted neuron, it is released from the heavy chain so that it can reach its active cleavage sites on the snare proteins. the light chain is released from the heavy chain by the reduction of the disulfide bond holding the two together. the reduction of this disulfide bond is mediated by the nadph-thioredoxin reductase-thioredoxin system. the light chain of bont acts as a metalloprotease on snare proteins that is dependent on zn(ii) ions, cleaving them and eliminating their function in exocytosis. there are 8 known isotypes of bont, bont/a – bont/h, each with different specific cleavage sites on snare proteins. snap25, a member of the snare protein family located in the membrane of cells, is cleaved by bont isotypes a, c, and e. the cleavage of snap-25 by these isotypes of bont greatly inhibits their function in forming the snare complex for fusion of vesicles to the synaptic membrane. bont/c also targets syntaxin-1, another snare protein located in the synaptic membrane. it degenerates these syntaxin proteins with a similar outcome as with snap-25. a third snare protein, synaptobrevin (vamp), is located on cell vesicles. vamp2 is targeted and cleaved by bont isotypes b, d, and f in synaptic neurons. the targets of these various isotypes of bont as well as tetanus neurotoxin (tent) are shown in the figure to the right. in each of these cases, botulinum neurotoxin causes functional damage to snare proteins, which has significant physiological and medical implications. by damaging snare proteins, the toxin prevents synaptic vesicles from fusing to the synaptic membrane and releasing their neurotransmitters into the synaptic cleft. with the inhibition of neurotransmitter release into the synaptic cleft, action potentials cannot be propagated to stimulate muscle cells. this result in paralysis of those infected and in serious cases, it can cause death. although the effects of botulinum neurotoxin can be fatal, it has also been used as a therapeutic agent in medical and cosmetic treatments. tetanus neurotoxin the breakdown of responsibilities and mechanisms of the heavy (hc) and light chain (lc) of tetanus neurotoxin: the hc assists in binding of tent to both the ganglioside receptor and the final receptor. once tent is in the vesicle in the inhibitory interneuron space the hc assists in translocation of the lc into the cytoplasm. then the lc, characterized by zinc endopeptidase activity, inhibits neurotransmission by cleavage of synaptobrevin 1. tetanus toxin, or tent, is composed of a heavy chain (100kda) and a light chain (50kda) connected by a disulfide bond. the heavy chain is responsible for neurospecific binding of tent to the nerve terminal membrane, endocytosis of the toxin, and translocation of the light chain into the cytosol. the light chain has zinc-dependent endopeptidase or more specifically matrix metalloproteinase (mmp) activity through which cleaveage of synaptobrevin or vamp is carried out. for the light chain of tent to be activated one atom of zinc must be bound to every molecule of toxin. when zinc is bound reduction of the disulfide bond will be carried out primarily via the nadph-thioredoxin reductase-thioredoxin redox system. then the light chain is free to cleave the gln76-phe77 bond of synaptobrevin. cleavage of synaptobrevin affects the stability of the snare core by restricting it from entering the low energy conformation which is the target for nsf binding. this cleavage of synaptobrevin is the final target of tent and even in low doses the neurotoxin will inhibit neurotransmitter exocytosis. role in neurotransmitter release neurotransmitters are stored in readily releasable pools of vesicles confined within the presynaptic terminal. during neurosecretion/exocytosis, snares play a crucial role in vesicle docking, priming, fusion, and synchronization of neurotransmitter release into the synaptic cleft. the first step in synaptic vesicle fusion is tethering, where the vesicles are translocated from the reserve pool into physical contact with the membrane. at the membrane, munc-18 is initially bound to syntaxin 1a in a closed structure. it is postulated that the dissociation of munc-18 from the complex frees syntaxin 1a to bind with the v-snare proteins. the next step in release is the docking of vesicles, where the v- and t-snare proteins transiently associate in a calcium-independent manner. the vesicles are then primed, wherein the snare motifs form a stable interaction between the vesicle and membrane. complexins stabilize the primed snare-complex rendering the vesicles ready for rapid exocytosis. the span of presynaptic membrane containing the primed vesicles and dense collection of snare proteins is referred to as the active zone. voltage-gated calcium channels are highly concentrated around active zones and open in response to membrane depolarization at the synapse. the influx of calcium is sensed by synaptotagmin 1, which in turn dislodges complexin protein and allows the vesicle to fuse with the presynaptic membrane to release neurotransmitter. it has also been shown that the voltage-gated calcium channels directly interact with the t-snares syntaxin 1a and snap-25, as well as with synaptotagmin 1. the interactions are able to inhibit calcium channel activity as well as tightly aggregate the molecules around the release site. there have been many clinical cases that link snare genes with neural disorders. deficiency in snap-25 mrna has been observed in hippocampal tissue of some schizophrenic patients, a snap-25 single-nucleotide polymorphism is linked to hyperactivity in autism-spectrum disorders, and overexpression of snap-25b leads to the early onset of bipolar disorder. role in autophagy macroautophagy is a catabolic process involving the formation of double-membrane bound organelles called autophagosomes, which aid in degradation of cellular components through fusion with lysosomes. during autophagy, portions of the cytoplasm are engulfed by a cup-shaped double-membrane structure called a phagophore and eventually become the contents of the fully assembled autophagosome. autophagosome biogenesis requires the initiation and growth of phagophores, a process that was once thought to occur through de novo addition of lipids. however, recent evidence suggests that the lipids that contribute to the growing phagophores originate from numerous sources of membrane, including endoplasmic reticulum, golgi, plasma membrane, and mitochondria. snares play important roles in mediating vesicle fusion during phagophore initiation and expansion as well as autophagosome-lysosome fusion in the later stages of autophagy. though the mechanism of phagophore initiation in mammals is unknown, snares have been implicated in phagophore formation through homotypic fusion of small, clathrin-coated, single-membrane vesicles containing atg16l,