Latrotoxin and Bungarotoxin Notes

Latrotoxin and Bungarotoxin

Pills and Poisons - NE2516 Cellular Neuroscience

• Dr. Stuart Greenhill (s.greenhill@aston.ac.uk)

Previously on Pills and Poisons

• Review of synaptic vesicle fusion and the role of Botulinum toxins (BoTX) in disrupting this process.

  • Different BoTX serotypes (A, B, C, D, E, F, G) and tetanus toxin (TeTX) target specific SNARE proteins (Synaptobrevin, SNAP-25, Syntaxin) essential for vesicle fusion.

Evoked vs. Spontaneous Synaptic Vesicle Fusion

• Evoked Fusion:

  • Involves Synaptobrevin 2, Syntaxin 1, SNAP25, Synaptotagmin 1, and the SNARE complex.

  • Calcium $Ca^{2+}$ is crucial.

• Spontaneous Fusion:

  • Involves Synaptobrevin 2, VTI1A, VAMP7 (or others), SNAP25 (or others), Syntaxin 1 (or others), Complexin, DOC2, and leads to fusion pore formation.

Botox and Tetanus Toxin Mechanism

• Botox and tetanus toxins hijack vesicles to enter neurons.

• Process:

  1. Toxin binds to receptors on the cell surface.

  2. Endocytosis of the toxin.

  3. The toxin is transported to an endosome.

  4. The toxin exits the endosome and enters the cytosol.

• Differences:

  • Botox (BoNT) primarily affects motoneurons at the neuromuscular junction (NMJ).

  • Tetanus toxin (TeNT) affects inhibitory interneurons in the central nervous system (CNS).

• Exploitation of pH gradient by Tetanus toxin: Molten globules making pores for their own use

Bungarotoxins

• Found in Krait snake venom.

• Neurotoxins with presynaptic and postsynaptic actions.

  • Beta-bungarotoxin: Presynaptic action.

  • Alpha-bungarotoxin: Postsynaptic action (also Kappa-bungarotoxin).

Beta-Bungarotoxin

• Inhibits acetylcholine (ACh) release through multiple mechanisms:

  1. Affects synaptic vesicular proteins like SNAREs.

  2. Inhibits presynaptic calcium channels.

  3. Exhibits Phospholipase A2 (PLA2) activity.

Phospholipase A2 (PLA2) Venoms

• PLA2 enzymes in snake venom have neurotoxic effects (presynaptic).

• Mechanism:

  • PLA2 hydrolyzes phospholipids, producing free fatty acids (like arachidonic acid) and lysophospholipids.

  • Arachidonic acid leads to the production of prostaglandins, thromboxanes, and leukotrienes, causing inflammation, hemorrhage, cytotoxicity, and myotoxicity.
    *Venoms contain PLA2 and can cause:

  • Neurotoxicity

  • Hemolysis

*Snake venom PLA2 chemical structure is represented as:

$\text{CH}<em>2 \text{-O-C-R}</em>1$
$|$
$\text{CH-O-C-R}<em>2$ $||$ $\text{O}$ $\text{CH}</em>2 \text{-O-PO-R}_3$

• Pharmacological Effects:

  • Alter the environment of target proteins.

  • Anticoagulant effects.

  • Platelet aggregation (initiation/inhibition).

  • Edema.

  • Convulsant/hypotensive activity.

  • Organ tissue damage (liver, kidney, lungs).

Beta-Bungarotoxin Mechanism of Action

• Research paper: Mechanism of Action of β-Bungarotoxin, a Presynaptically Acting Phospholipase A2 Neurotoxin: Its Effect on Protein Phosphorylation in Rat Brain Synaptosomes.

• Key Findings:

  • No inhibition of ATP synthesis.

  • No stimulation of phosphatase 1, 2A, or calcineurin.

  • Effects not secondary to arachidonic acid or its metabolites.

  • No effect on subcellular calmodulin distribution.

  • No direct inhibition of cAMP-kinase, Ca2+/calmodulin-kinase II, or protein kinase C.

  • May modify substrates or their accessibility to kinases.

  • Inhibition of MARCKS phosphorylation by endogenous (but not exogenous) protein kinase C.

  • May interfere with the translocation of kinases or substrates between the cytosol and plasma membranes.

Beta-Bungarotoxin Structure & Function

• Has an 'A' chain and a 'B' chain, each with distinct effects.

  • A-chain: PLA-like effects.

  • B-chain: Induces apoptosis.

• Significant action involves phosphorylation of MARCKS protein.

MARCKS Protein

• MARCKS: Myristoylated Alanine-Rich C Kinase Substrate.

• An intrinsically disordered protein.

• Functions:

  • Crosslinks actin filaments at the plasma membrane (reduced upon PKC phosphorylation or Ca2+/calmodulin binding).

  • Concentrates signaling molecules like PIP2 within membrane microdomains.

  • Modulates signal transduction (e.g., PLCγ-catalyzed production of inositol trisphosphate (IP3) and diacylglycerol (DAG) from PIP2).

  • Interacts with PSA-modified proteins, such as neural cell adhesion molecule (NCAM), facilitating cell-cell interactions.

  • Facilitates docking and fusion of Rab10-positive vesicles.

• In the synapse, MARCKS contributes to learning and memory.

Alpha-Bungarotoxin

• Binds competitively, rapidly, and almost irreversibly to the nicotinic acetylcholine receptor at the neuromuscular junction.

• Used as a tracking tool due to its high binding affinity.

Latrotoxins (Black Widow Spider Venom)

• Black widow spider venom contains multiple latrotoxins.

  • Alpha-latrotoxin targets vertebrates.

  • Five latroinsectotoxins.

  • Latrocrustatoxin.

• C. elegans can survive in concentrations 1000 times the lethal human dose of alpha-latrotoxin.

Latrotoxin Structure and Function

• Exists as dimers or tetramers.

• Tetramer: Inserts into the membrane, forming pores that allow calcium influx.

• Dimer: Binds to and signals via latrophilin (aka CIRL - Calcium-Independent Receptor of Latrotoxin).

Latrotoxin Mechanism

• Latrotoxin (LTX) interacts with nerve terminals and synaptic vesicles.

• Calcium influx is crucial for its action.

• Binds to Neurexin and Latrophilin. *Depending on presence of Calcium ions, different Latrotoxin oligomers engage different interaction with the cell:

  • Calcium present, LTX 2x interaction, leading to signaling

  • Calcium absent, LTX 4x interaction, leading to membrane insertion

Latrophilin

• A type of Adhesion GPCR (G protein-coupled receptor).

• Function not fully understood, but thought to be involved in tactile sensation.

• Latrophilin 3 is essential for Schaffer synapse formation.

• Errors in synaptogenesis can lead to diseases or conditions.

Studying Latrotoxins

• Mutant alpha-latrotoxin (unable to form pores) can be used to increase neurotransmitter release during experiments.

• Allows separation of different toxin actions and investigation of receptors.

Readings

• Relevant research articles on latrotoxins and their receptors.

  • Alpha-Latrotoxin and Its Receptors (Ushkaryov, Rohou, and Sugita).

  • Presynaptic neurotoxins: An expanding array of natural and modified molecules (Davletova, Ferrari, Ushkaryov).

  • Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins (Sando, Jiang, Südhof).

  • The multiple actions of black widow spider toxins and their selective use in neurosecretion studies (Ushkaryov, Volynski, Ashton).