VAMP2 Human
Description
Functional Roles in Membrane Fusion
VAMP2 mediates synaptic vesicle fusion through the following mechanisms:
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SNARE complex formation: VAMP2 binds syntaxin-1A and SNAP-25 to form a four-helix bundle, generating mechanical force for membrane fusion .
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Lipid raft interactions: Cholesterol-rich lipid raft regions weaken VAMP2-membrane binding, freeing the SNARE motif for complex assembly .
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Neurotransmitter release: VAMP2 depletion reduces spontaneous and evoked synaptic transmission by 90%, highlighting its necessity .
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Antibody secretion: In plasma cells, VAMP2 partners with SNAP23 and STX4 to facilitate antibody exocytosis .
Neurodevelopmental Disorders
Heterozygous VAMP2 mutations cause neurodevelopmental disorder with hypotonia and autistic features (NEDHAHM). Key findings include:
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Pathogenic variants: Truncating (e.g., p.Arg56X) and missense (e.g., p.Arg56Leu) mutations impair vesicle fusion, leading to global developmental delay, epilepsy, and autism .
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Electrophysiological deficits: Mutations reduce synaptic vesicle recycling and neurotransmitter release, as shown in neuronal models .
Biomarker Potential in Dementia
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Discriminatory expression: Elevated VAMP2 mRNA in peripheral blood mononuclear cells distinguishes mixed dementia from Alzheimer’s disease (AUC = 82%) .
Key Research Findings
Table 2: Select Studies on VAMP2
Therapeutic Insights
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Aminopyridine treatment: Enhances exocytosis in neurons with VAMP2 mutations by prolonging action potentials .
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Gene-environment interactions: Lipid composition modulation (e.g., cholesterol levels) may rescue VAMP2 dysfunction .
Future Directions
Product Specs
Q&A
What is the structure and function of VAMP2 in humans?
VAMP2 is a core component of the SNARE complex that mediates fusion of synaptic vesicles to release neurotransmitters. Upon interaction with other SNAREs, VAMP2 transforms from a largely unstructured state to having four distinct structural domains: a proline-rich N-terminal domain (residues 1-30), a SNARE motif/core domain (residues 31-85), a juxtamembrane domain (JMD, residues 86-95), and a C-terminal transmembrane domain (TMD, residues 96-114) . This transformation from disordered to ordered state releases free energy that drives SNARE complex formation, highlighting the conformational diversity of synaptic SNARE proteins . VAMP2 plays a critical role in cell communication in the brain and is involved in regulated exocytosis in neurons and endocrine cells . The protein forms helix bundles with syntaxin-1A and SNAP-25 to produce the force necessary for fusion pore formation, ultimately mediating vesicle fusion and neurotransmitter release .
Research methodology: Structural studies of VAMP2 typically employ techniques such as in-cell NMR spectroscopy to observe VAMP2 conformational changes in mammalian cells, particularly during interactions with lipid membranes and other SNARE proteins. Functional assays often use genetic knockout and rescue experiments in neuronal cell cultures to assess the specific contributions of VAMP2 to vesicle fusion events.
How does membrane environment regulate VAMP2 function?
VAMP2 exhibits different conformations in various membrane regions, which significantly impacts its function. Mass-spectrometry-based lipidomic profiling of synaptic vesicle membranes has revealed that the membrane environment directly influences VAMP2 structure and activity . Notably, cholesterol-rich lipid raft regions markedly weaken the membrane association of the VAMP2 SNARE motif, which releases the motif and facilitates SNARE assembly . This contrasts with non-raft regions where VAMP2 shows stronger membrane association. The dynamic regulation by membrane composition provides spatial control of SNARE assembly.
Research methodology: To study membrane effects on VAMP2, researchers can manipulate cellular cholesterol levels and analyze VAMP2 conformations using in-cell NMR spectroscopy . Lipidomic profiling of synaptic vesicle membranes using mass spectrometry helps identify specific lipid compositions that influence VAMP2 behavior. These approaches allow researchers to understand how different membrane regions regulate VAMP2 structure and function.
What are the expression patterns and localization of VAMP2?
VAMP2 primarily resides on synaptic vesicle membranes in neurons, positioning it strategically for its role in neurotransmitter release . It is highly expressed in neuronal and neuroendocrine cells, such as PC12 cells, which serve as valuable models for studying VAMP2 trafficking and function . Within cells, VAMP2 shows differential localization in distinct membrane regions, adopting specific conformations based on the local lipid environment .
Research methodology: Cellular localization of VAMP2 can be studied using immunofluorescence microscopy with VAMP2-specific antibodies or fluorescently tagged VAMP2 constructs. Subcellular fractionation followed by Western blotting can quantify VAMP2 abundance in different cellular compartments. Single-cell RNA sequencing and tissue-specific transcriptomics help map VAMP2 expression patterns across different cell types and brain regions.
What experimental models are optimal for studying human VAMP2 function?
Several experimental systems have been established for investigating VAMP2 function in contexts relevant to human biology:
| Model System | Applications | Advantages | Limitations |
|---|---|---|---|
| PC12 cells | Trafficking, exocytosis | Endogenous VAMP2 expression, neuroendocrine properties | Not primary neurons |
| HEK293 cells | Single vesicle fusion visualization | Amenable to high-resolution imaging | Non-neuronal background |
| Primary rat striatal neurons | Physiological VAMP2 function | Native neuronal environment | Species differences from human |
| VAMP2 knockout neurons | Rescue experiments | Clean background for functional studies | Compensatory mechanisms |
PC12 cells (rat neuroendocrine cell line) endogenously express high levels of VAMP2 and serve as excellent models for studying VAMP2 trafficking and function, with mechanistic insights from these cells having previously translated well to neurons and animal models . HEK293 cells provide a system for visualizing individual fusion events of vesicles containing VAMP2 and various cargo molecules . Primary rat striatal neurons offer a physiologically relevant context for studying VAMP2-mediated neurotransmitter release . VAMP2 knockout neurons are valuable for studying the effects of VAMP2 deficiency and for rescue experiments with mutant forms of VAMP2 .
Research methodology: When selecting a model system, researchers should consider whether they need to study endogenous VAMP2 function (primary neurons, PC12 cells) or whether heterologous expression is suitable (HEK293 cells). For genetic manipulation, CRISPR-Cas9 editing of VAMP2 or doxycycline-inducible shRNA systems allow for controlled knockdown or knockout studies .
How can VAMP2 trafficking be visualized in living cells?
Advanced imaging techniques have revolutionized our ability to visualize VAMP2 dynamics during vesicle fusion events:
High-resolution multichannel total internal reflection fluorescence microscopy (TIR-FM) enables direct visualization of individual plasma membrane fusion events containing VAMP2 . This approach can be enhanced by using pH-sensitive GFP mutants (super ecliptic pHluorin, SpH) tagged to the extracellular terminus of cargo molecules, as SpH fluorescence is quenched in acidic compartments but increases when exposed to the neutral extracellular environment during fusion events . VAMP2-pHuji constructs provide similar capabilities for visualizing VAMP2 localization during fusion .
Research methodology: For simultaneous visualization of VAMP2 and specific cargo proteins, researchers can design dual-color imaging experiments using spectrally distinct fluorophores (e.g., VAMP2-pHuji and SpH-tagged receptors) . TIR-FM microscopy with high-speed multichannel acquisition (>10 frames per second) is essential for capturing rapid fusion events. Analysis should include quantification of colocalization between VAMP2 and cargo signals, as well as temporal dynamics of fluorescence changes during fusion events.
What is the evidence for VAMP2's cargo selectivity in vesicle trafficking?
Recent research has revealed that VAMP2 exhibits surprising selectivity in the cargo it transports, challenging the previous view of v-SNAREs as general mediators of vesicle fusion:
High-speed multichannel imaging studies have demonstrated that VAMP2 is preferentially enriched in vesicles that mediate the surface delivery of μ opioid receptor (MOR), but not other cargos like β2 adrenergic receptor (B2AR) or transferrin receptor (TfR) . Interestingly, VAMP2 does not show preferential localization on MOR-containing endosomes, suggesting that v-SNAREs are copackaged with specific cargo into separate vesicles from the same endosomes . Depletion of endogenous VAMP2 using inducible shRNA significantly reduced the rates of recycling of MOR in PC12 cells and rat striatal neurons, confirming VAMP2's selective role in MOR trafficking .
Research methodology: To investigate cargo selectivity, researchers can employ simultaneous imaging of multiple fluorescently tagged cargoes alongside VAMP2 during vesicle fusion events. Quantitative analysis of colocalization frequencies and selective knockdown/knockout experiments can validate functional relationships between VAMP2 and specific cargo proteins.
How do VAMP2 mutations contribute to human neurodevelopmental disorders?
VAMP2 mutations have been implicated in a recently identified neurodevelopmental syndrome:
VAMP2-related syndrome (also called neurodevelopmental disorder with hypotonia and autistic features with or without hyperkinetic movements) is caused by pathogenic variants in the VAMP2 gene . Five heterozygous de novo mutations affecting the C terminus of the VAMP2 SNARE motif have been identified in individuals with this disorder, including two single-amino-acid deletions and three non-synonymous variants (such as p.Ser75Pro and p.Phe77Ser) . Clinical features include intellectual disability, developmental delay, hypotonia present since birth, autistic features, choreic movements, and severe language impairment . Brain imaging in affected individuals may show abnormalities such as thin corpus callosum and delayed myelination .
Research methodology: Whole exome sequencing (WES) of patient-parent trios is the primary method for identifying de novo VAMP2 mutations . Functional characterization of these mutations can be performed using in vitro SNARE complex assembly assays, vesicle fusion assays in reconstituted systems, and rescue experiments in VAMP2-deficient neurons. Patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons represent a valuable model for studying the effects of VAMP2 mutations in a human genetic background.
What approaches can characterize VAMP2 structural changes during SNARE complex assembly?
Understanding the structural transitions of VAMP2 during SNARE complex formation is critical for elucidating the molecular mechanisms of vesicle fusion:
In-cell NMR spectroscopy provides insights into the dynamic membrane association of the VAMP2 SNARE motif and its structural changes in mammalian cells . This technique has revealed that VAMP2 is largely unstructured in the absence of interaction partners but adopts a more stable structure upon interaction with other SNAREs . The transformation from a disordered to an ordered state releases free energy that contributes to driving SNARE complex formation and membrane fusion .
Research methodology: Researchers investigating VAMP2 structural dynamics can employ a combination of in-cell NMR spectroscopy, single-molecule Förster resonance energy transfer (FRET), and molecular dynamics simulations. Site-directed spin labeling coupled with electron paramagnetic resonance (EPR) spectroscopy can provide information about specific conformational changes in VAMP2 during interactions with other SNARE proteins and membranes.
Product Science Overview
Synaptobrevin-2 is characterized by its proline-rich N-terminal part, a highly conserved hydrophilic domain, a transmembrane anchor, and a C-terminal . It is one of the key proteins involved in the formation of the SNARE complex, which consists of four α-helices: one contributed by synaptobrevin, one by syntaxin, and two by SNAP-25 (Synaptosome-Associated Protein 25) .
The primary function of Synaptobrevin-2 is to mediate the fusion of synaptic vesicles with the plasma membrane, facilitating the release of neurotransmitters into the synaptic cleft. This process is triggered by an influx of calcium ions (Ca²⁺) into the nerve terminal following an action potential .
During synaptic vesicle fusion, Synaptobrevin-2 forms a complex with the plasma membrane SNARE proteins, syntaxin 1 and SNAP-25. This complex brings the vesicle and plasma membranes into close proximity, allowing them to fuse and release their contents . The tight coupling of the SNARE motif to the transmembrane region of Synaptobrevin-2 is crucial for this process, ensuring efficient Ca²⁺-triggered exocytosis .
Synaptobrevin-2 is targeted by various bacterial toxins, such as tetanospasmin from Clostridium tetani, which causes tetanus, and botulinum toxin from Clostridium botulinum, which causes botulism. These toxins cleave Synaptobrevin-2, disrupting neurotransmitter release and leading to severe neurological symptoms .
Human recombinant Synaptobrevin-2 is produced using recombinant DNA technology, which involves inserting the gene encoding Synaptobrevin-2 into a suitable expression system, such as bacteria or yeast. This allows for the production of large quantities of the protein for research and therapeutic purposes .
