Mechanism of Action

 

SNARE mechanism

Rothman suggested that the ATPases NSF drives membrane fusion by hydrolysing ATP however this is now known to be incorrect. NSF is now known to dissociate the “cis” SNARE complex formed after fusion has occurred when SNAREs lie in the target membrane, dissociation allows the SNAREs contributed from the vesicle to be recycled back to the compartment the vesicle originated from. There is plenty of evidence to indicate that SNAREs facilitate membrane fusion:

 

• Protein-protein interaction reproduction in test tube –by adding combinations of proteins together it was shown which proteins could to bind to one another to demonstrate which components made up the fusion machinery.
• Yeast secretory mutants – identified the genes required in the secretory pathway, so that proteins involved in membrane fusion could be identified.
• Model Organism- Drosophilia and C.elegans, can create temperature sensitive mutants. Difficult to make a knock out which isn’t temperature sensitive because if the protein is essential for fusion then the organism will die before maturation. C. elegans was used to investigate unc proteins whose homologs in mammals are munc proteins. Essentially when SNAREs are knocked out there is no fusion indicating they are essential, whereas when NSF is knocked out there is fusion to begin with but it stops because the SNAREs cannot be reused.
• Patch clamping and high resolution imaging.
• ATP hydrolysis is insufficient to drive membrane fusion – shown by a number of experiments including tetanus and botulism toxin studies.
• Tetanus and Botulism toxins – these toxins target the SNAREs involved in neuroexocytosis, they cleave the toxins so that they cannot function. Experiments utilising these toxin showed that when the SNAREs were cleaved membrane fusion did not occur. In addition in the absence of toxins when NSF action was prevented fusion did occur. This strongly suggests that NSF does not have a direct role in fusion whereas SNAREs do.

 

There are now several models for how SNAREs drive membrane fusion, one suggestion is that SNARE assembly underlies fusion by generating mechanical force. This is the widely accepted but still controversial view. Two models that are based on this include:

 

1) Zipper model 

This model suggest that the SNARE proteins on the opposing membranes bind to one another forming a “trans” SNARE complex by zippering of the alpha helices from the N-terminus to the C-terminus (which is the transmembrane segment of the SNARE) to form a “trans” SNARE complex. The zippering action is believed to create mechanical force that bends the membrane to overcome the energy required for fusion. So this model suggests that mechanical force directly causes membrane fusion.

 

2) Stalk hypothesis

This model for membrane fusion describes fusion as a series of lipid transition states of the membrane. Suggesting that the membrane proceeds through a series of intermediates until the fusion pore forms, assuming the membrane is similar to a bendable sheet ignoring any movement of the lipids in the membrane. According to this model there are rigid linkers between the transmembrane domain and the SNARE motif that forms the helix, as a result of the stiff linkers mechanical energy is transferred from the alpha helices formed from the SNARE motif to the membrane which deforms the membrane first causing curvature leading to hemifusion states. Hemifusion is defined as a state in which the outer membrane leaflets are continuous but no aqueous pore has formed. The first hemifusion state forms the fusion stalk and the second hemifusion state is characterised by the formation of transmembrane contact. Following these states the fusion pore forms.

Evidence

There is variable evidence for how many SNARE complexes would be required to generate enough mechanical force to cause membrane curvature. Whether SNAREs form the fusion pore is contentious issue, several papers in the additional reading covers the arguments around this topic.

Perturbtion of hydrophilic-hydrophobic boundary of the membrane is the alternative explanation to mechanical force as the cause of fusion. This model suggests that the amphillic region of the SNAREs could disrupt the hydrophillic-hydrophobic binding in the membrane making the membrane able to bend to allow fusion. 

Evidence

This model is supported by in vitro studies that showed isolated SNARE components could drive fusion of liposomes.

Despite advantages and disadvantages of the models ALL models all fail to address why only QabcR SNARE complexes are fusion compatible?

 

Role of SNAREs in regulated secretion.

There are two types of secretion regulated and constitutive, constitutive occurs continuously whereas regulated only occurs when required. Regulated secretion tends to be found in cells that have to secrete a substance rapidly (hormones, enzymes, neurotransmitters, immune system chemical mediators) such a neurons releasing neurotransmitter. Why is it important to understand regulated secretion? Unregulated secretion of a product that is normally regulated has been implicated in a number of diseases including:

 

• Endocrine disorders – for example Cushing’s syndrome.
• Allergen disorders – for example Asthma.
• Immune disorders – for example Irritable bowel syndrome(IBS).
• Metabolic disorders – examples include type II Diabetes and obesity.
• Mood dosorders – such as depression.

 

The host of ailments caused by lack of regulation highlight its importance. MIscroscopy coupled with GFP expression on vesicles was used to visualise cells the undergo regulated secretion and other cells that undergo constitutive secretion. By this method it was shown that cells that underwent regulated secretion had immobilised vesicles docked at the plasma membrane that were only released on signal. Whereas cells that displayed constitutive secretion did not have the docked pool of vesicles. Therefore the cells that display regulated secretion must have specialised fusion machinery to have a docked and immobilised pool of vesicles. What is the role of SNAREs in regulated secretion of neurons?

 

Neurotransmission occurs at the synapse between neurons in the central nervous system(CNS) or at the neuromuscular junction in the peripheral nervous system(PNS). The video at the bottom of the page gives a brief description of how neurotransmission occurs at a chemical synapse

 

In neurons there are two types of storage vesicles for neurotransmitters to be released. Dense core vesicles (DCV)/secretory granules store peptides and catecholamines and synaptic vesicles store classical neurotransmitters. The CNS tends to utilise synaptic vesicles and the PNS uses both types.DCV are produced from the golgi in the soma of the neuron and then are transported along the axon to the presynaptic terminal. Transport occurs along cytoskeleton with the aid of molecular motors( kinesin and dyenin).

 

Studies have shown that DCV matures at the pre-synaptic terminal. The contents become more condensed and lumen of the vesicle acidifies, excess membrane is removed and the clathrin coat is lost from the vesicle. In dense core vesicles processing of the peptides can occur in the vesicle,for example lots of hormones are initially produced in a pro-form that is cleaved to yield the mature hormone. Processes governing how a neurotransmitter is selectively packaged in a vesicle is still not understood in detail. But studies suggest that the information to cause packaging into vesicles is contained in the protein rather than the vesicle. A major difference between DCV and synaptic vesicles is that the DCV cannot be refilled to be recycled.

 

In contrast to DCV synaptic vesicles are produced locally from the endosome at the synapse and are more rapidly released and recycled. It is known that the vesicles pass through different stages in “vesicle cycle” including:

 

1. Loading of the vesicle.
2. Storage of the vesicle.
3. Mobilisation of the stored vesicle to become docked.
4. Primed vesicles ready for fusion.
5. Calcium sensing to initiate fusion.
6. After fusion recycling of vesicle

By amperometry measuring membrane capacitance change upon flash photolysis in chromaffin cells three phases of vesicles release were observed:

1. Readily releasable pool (RRP) – additional primed from the SRR and released immediately after stimulation.
2. Slowly releasable pool (SRP) – require additional priming step before they can be released. Once the RRP pool is depleted these are primed and released.
3. Unprimed pool (UPP) – docked but cannot undergo fusion as not primed. When the SRP pool is depleted then these are primed to be released but this takes time and so is rate limiting.

Research showed different pools of vesicles. How are the separate pools achieved?

Research by Sorensen into mutants of C.elegans the nematode worm revealed that specific proteins regulate docking and fusion of vesicles by interacting with SNAREs. The mutants of C.elegans had abnormal synaptic transmission and as a result displayed movement disorders and so were named uncoordinated which was shortened to Unc. By microscopy these mutants were shown to have a complete lack of docked vesicles and they vesicles present were unable to fuse. Therefore it was suggested that the protein encoded by the mutated Unc gene was responsible for docking and fusion. It was shown that the Unc protein encoded by the Unc could bind to syntaxin one of the SNAREs of the fusion machinery and alter its conformation. Normally syntaxin would be in the closed configuration and so unable to bind to other SNAREs, but when Unc bound the syntaxin underwent a conformational change to the open configuration so that it could bind to other SNAREs. Therefore Unc proteins s allow formation of vesicles docked to the membrane by the formation of SNARE complexes. So that when calcium levels increase fusion can proceed once the calcium sensor synaptotagmin detects the rise in calcium.

The same mechanism is thought to operate in mammals as homologs to the Unc proteins called Munc proteins have been discovered.

Although the model suggested above gives some information on how vesicles become docked in regulated secretion its believed thatadditional proteins regulated this process.

 

 The video above gives a brief overview of synaptic transmission, which was where SNAREs were first characterised.