NESC 2570 Lecture Notes - Lecture 7: C2 Domain, Syntaxin, Vesicle Fusion
27 Jun 2018
School
Department
Course
Professor
Exocytosis
Overview: Transmitter exocytosis requires priming and calcium-dependent fusion of synaptic vesicles
○
Proteins involved in synaptic vesicle fusion with the plasma membrane
The SNARE complex brings synaptic vesicle membrane and plasma membrane into close
proximity
○
Clostridial neurotoxins inhibit transmitter release by cleaving SNAREs
○
Potential role of the SNARE complex in membrane fusion
○
Munc18/nSec1 may regulate SNARE complex assembly and facilitate membrane fusion
○
NSF and Ĵ-SNAP are involved in the disassembly of SNARE complexes
○
Summary: The cycle of SNARE complex assembly and disassembly The calcium sensor for
synaptic vesicle fusion
○
Synaptotagmin I is a calcium- and phospholipid-binding protein
○
Evidence that synaptotagmin is the calcium sensor responsible for calcium-dependent fast
transmitter release.
○
Hypothetical mechanism of synaptotagmin action
○
○
Firstly, synaptic vesicles need to make it to the plasma membrane (this is called synaptic vesicle
docking)
○
Secondly, the vesicles have to be tethered to the plasma membrane - there are protiens that are specially
required to do this. This is called synaptic vesicle priming, only then can the vesicles fuse with the
membrane once calcium rises from the pre synaptic specialization
○
Transmitter Exocytosis Requires Priming and Calcium-Dependent Fusion of Synaptic Vesicles
During priming, the synaptic vesicle membrane and plasma membrane are brought into close proximity
through interactions of synaptic vesicle membrane proteins and plasma membrane proteins.
They have to make it to the synaptic vesicle and tethered to the plasma membrane which is called
synaptic vesicle priming
○
Priming requires 3 or 4 proteins called SNARE proteins (Snap Receptors)
○
○
Cooperative calcium binding to the calcium sensor leads to fusion of the closely apposed membranes
○
The SNARE Complex Brings Synaptic Vesicle Membrane and Plasma Membrane Into Close Proximity
Membrane fusion generally involves SNAREs (SNAP receptors): Proteins with 70-aa residue SNARE
motifs.
They are proteins that have long alpha helical structures - they are protein structures that look like
springs. They are coiled and they have 3 or 4 SNARES
○
There are fatty acids that help it stick to the plasma membrane
○
These alpha helices like to form a bundle - they are energetically favourable so it is hard to pull
them apart again
○
These two membranes are negatively charged (phospholipids) the two membranes will then repel
each other - the energy required for this is taken from the formation of the SNARE complex
○
○
Three or four SNARE proteins on the two fusing membranes form a SNARE complex: Assembly of
SNARE motifs into a parallel four-helical bundle.
○
Synaptic vesicle exocytosis is mediated SNAREs syntaxin and SNAP-25 (plasma membrane), and
synaptobrevin (SV membrane).
○
The SNARE complex is assembled by formation of a helical bundle of one SNARE motif of each
synaptobrevin and syntaxin and two SNARE motifs of SNAP-25.
○
Synaptobrevin resides in the synaptic vesicle membrane
○
Syntaxin resides in the plasma membrane
○
SNAP 25 is associated with the plasma membrane, there are fatty acids attached to the peptide which
allows it to stick to the plasma membrane
○
These three proteins together can form a complex
Alphahelicies can form coils and are energetically favourable which makes it very hard to pull
them away from each other
○
They are negatively charged so the two membranes repel each other, the force that pushes them
together is generated from the formation of the SNARE complex
○
○
Clostridal Neurotoxins Inhibit Neurotransmitter Release By Cleaving SNARE Proteins
Clostridial neurotoxins responsible for tetanus and botulism are highly specific proteases that block
neurotransmitter release by cleaving SNAREs
○
Inhibition of neurotransmitter release by botulinum toxins and tetanus toxin shows that SNARE
complex formation is required for synaptic vesicle fusion
○
Bacteria that have toxins that paralyze neurons in the CNS - these toxins can be fatal
Tetanus and botulism are caused by this
○
However, if you inject these toxins, the neuromuscular junction between motor neurons in your
skin and the smooth muscle cells become paralyzed which gets rid of wrinkles
○
○
If these toxins are on the synapse, you get complete restriction of neurotransmitter release
○
They cleave the SNARE proteins and restrict release
○
Potential Role of the SNARE Complex in Membrane Fusion
Free energy gained during the formation of the SNARE complex (the four-helical bundle is
energetically far more stable than the unstructured SNARE motifs) may be utilized for the energetically
unfavorable membrane fusion.
○
SNAREs reconstituted in liposomes in vitro cause membrane mixing in the absence of any other
proteins: SNARE complex may be "minimal machinery" sufficient for membrane fusion.
○
SNARE complex, however, is not sufficient for kinetically efficient membrane fusion. Other proteins
are required to keep SNARE complexes in a position favorable for membrane fusion (a), or to
structurally organize several SNARE complexes that have to act cooperatively (b).
○
Munc18/nSec1 May Regulate SNARE Assembly and Facilitate Membrane Fusion
Munc18-1 (Ł nSeq1), is essential for neurotransmitter release: Genetic ablation causes complete
inhibition of both calcium-dependent and spontaneous neurotransmitter release.
○
SM proteins - Munc18 is a member of this group (Sec1/Munc18) proteins participate in all intracellular
membrane fusion reactions.
○
Two function have been proposed for Munc18-1:
Munc18-1 binds to syntaxin (protein in the plasma membrane) in its "closed" conformation and
(N-terminal domain folded back on the SNARE motif, inhibiting SNARE complex formation):
Munc18-1 may regulate syntaxin recruitment into SNARE complexes
○
Munc18-1 also binds tightly to syntaxin in SNARE complexes. Munc18-1 greatly enhances
fusion of synaptobrevin-containing liposomes with syntaxin and SNAP-25-containing liposomes:
Munc18-1 may be an essential part of the fusion machinery itself, facilitating SNARE
complexmediated fusion.
○
Munc 18 is necessary to form SNARE complexes on one synaptic vesicles - without it, it will
abolish all release
○
○
Lecture Notes:
SM proteins, in which Munc18 is a member of, has a dual function
They bind to syntaxin (which remember is in SNARE protein in the plasma membrane) and
then binds to SNAP 25 and synaptogravin but it can only do so if there is a conformational
change. In it's closed form, it folds back on itself and is unable to interact with SNAP 25 or
synaptogravin and it needs to be unfolded. Munc18, binds to syntaxin in its closed form, so
they believe it has a role in unfolding the complex
○
Munc18 can also bind to already assembled SNARE complexes, it may be necessary to
coordinate the coordination of several SNARE complexes in the vesicle
○
○
○
NSF and Ĵ-SNAP are involved in the disassembly of SNARE complexes
SNARE complexes need to be disassembled after fusion to allow reuse of the individual SNAREs
○
SNARE complexes, however, are extraordinarily stable. A chaparone (protein folding enzyme) is
needed to dissociate it
○
NSF is the cognate chaperone for SNARE complexes. It binds to the complex via an adapter protein, Ĵ-
SNAP. NSF then unwinds the Ĵ-helical bundle of the SNARE complex, using ATP as an energy source
○
SNARE is needed to bring the two membranes together and it is due to these two proteins it is hard to
bring back together again
Chaperon proteins fold and unfold proteins - it allows for formational changes using ATP or GTP
○
Chaperon proteins often come with adapter proteins, SNAP is exactly that.
SNAP binds to the SNARE complex
○
○
It then recruits NSF which unfold the complex - this allows us to reuse the three proteins
(Syntaxin, SNAP 25 and Synaptogravin)
○
○
The cycle of SNARE complex assembly and disassembly
Current model of SNARE complex assembly and disassembly:
○
Synaptotagmin I is a calcium- and phospholipid-binding protein
Synaptotagmin 1 or 2, they are associated with different genes, is an abundant synaptic vesicle
membrane protein composed of a short N-terminal intravesicular sequence, a single transmembrane
region, and two cytoplasmic C2 domains.
The domain extends into the cytoplasm
○
C2 domains are proteins that bind calcium and phospholipids which are an imperative aspect of
membranes
○
Calcium is positively charged, so the more positive charges you have on Synaptotagmin, the
higher the affinity for this protein
○
It also binds to the SNARE complex
○
○
Both C2 domains of synaptotagmin 1 bind calcium: The C2A domain has binding sites for 3 calcium
ions, the C2B domain 2 sites. Calcium binding is cooperative.
They interact with amino acids and bind calcium
○
○
Synaptotagmin C2 domains also bind to phospholipid membranes in a calcium-dependent and calcium
in a phospholipid-dependent manner. The affinity of synaptotagmin for calcium is low in the absence of
phospholipids and increases to 5-10 µM in their presence.
○
Synaptotagmin also binds to SNARE complexes in a partly calcium dependent manner
It does its function at the SNARE complex
○
○
Evidence that syanptotamin is the calcium sensor responsible for calcium dependent fast transmitter
release
In synaptotagmin I knockout mast fast neurotransmitter release is absent - because the synchronous
release uses a different calcium sensor
○
In contrast, spontaneous, action potential- and calcium-independent release is unchanged, showing that
the synaptic vesicle fusion mechanism itself is not affected.
○
Also, asynchronous transmitter release (red arrows in figure) is unaffected in synaptotagmin-/- mice.
Asynchronous release is caused by synaptic vesicle fusions that are triggered in a calcium-dependent
manner, but with some delay, and has to rely on a different calcium sensor than synaptotagmin.
It uses a different calcium sensory in central neurons and occurs miliseconds after the action
potential
○
○
Post synaptic currents occur after the stimulation which is called asynchronous release which occurs
immediately (1-3 ms) after the action potential while synchronous release occurs hundreds of
miliseconds after the action potential
○
Hypothetical Mechanism of Synaptotagmin Function
Synaptotagmin binds to the SNARE complex via a polybasic region in the C2B domain before Ca2+
influx
○
Calcium binding leads to additional electrostatic charges on the C2A and C2B domains, causing them
to bind negatively charged phospholipid membranes. The C2B domain has been shown to bind to two
different phospholipid bilayers, such that it likely aids in bringing synaptic vesicle membrane and
plasma membrane into very close apposition.
○
The close apposition of two negatively charged phospholipid bilayers is energetically unfavorable. This
promotes membrane fusion
○
When you have calcium influx, the domains (both C2A and C2B) gets positively charged which attracts
negative charges
Because both membranes are negatively charged (energetically unfavourable), the layers fuse
with each other to make a fusion pore
○
○
A kind of endocytosis that requires the construction
of a proteins lattice over the protein that needs to be
endocytosed. This is called nucleation.
The Lattice that is necessary for endocytosis needs to
be dissembled again.
For each synaptic vesicle that is exocytosed, you need competitor
endocytosis
So one vesicle will get endocytosed
○
○
Endocytosis occurs according to a mechanism called clatherin mediated
endocytosis which is a type of endocytosis that requires the formation of a
protein lattice over the membrane that will be endocytosed
This is called nucleation
○
○
Once the lattice is formed, the membrane graduates until you have a
vesicle stock. After the stock is formed, a protein complex arrives and
form around the stock and pinches off at the vesicle stock and can create a
new vesicle
○
Clathrin needs adapter proteins, one being AP-2 which binds to
synaptogravin. Once it has bound to proteins in the synaptic membrane, it
recruits another protein called AP180 which recruits clathrin
○
Nucleation requires the formation of this lattice made up of clatherin
Clatherin is made up of two proteins, a clatherin light chain
and a clatherin heavy chain
○
Two clatherin heavy chains will interact with three light
chains and polymerize
○
○
Dynamin pinches off the synaptic vesicles after polymerizing around
the stock (see photo). Actin is very important in the fission process
by helping dynamin pinch off the synaptic vesicle. Actin exists in
two forms, G Actin and F Actin. F actin makes long filaments
○
Adapter proteins allow clatherin to bind to the plasma membrane
○
AP-2 binds to many proteins that need to be endocytosed
Once it binds, it will recruit another adapter protein called AP180
Clatherin nucleation cannot occur without the binding of
AP180 to AP2
§
○
○
Hsc70 is another adaptor protein that assembles the AP-2 and AP180
complex for clathrin. It needs auxilin which is an adaptor protein to make
the lattice.
○
Dynamin can polymermarize and will pinch off the synaptic vesicles from the stock
○
Can't have any more endocytosis than exocytosis or else your plasma membrane will shrink, so
they must be tightly linked. One concept is the regulation of phsophatidylinositol which are
lipids that have a sugar that can have phosphate groups themselves. The lipids act as second
messengers.
○
Actin is important in the fission process by helping dynamin to pinch off the vesicles
○
Actin exists as G Actin and F actin, G actin is a monomer while F actin is a polymer
F actin makes very long filaments
○
○
Hsc70 is also a chaporon protein and dissasembles the AP-2 and AP180 complex
It needs auxilin to bind to the clatherin lattice
○
○
Endocytosis and exocytosis are tightly linked through phosphatidylinositides
They have fatty acids and glyercol, and a sugar called nocetol which can have
phosphate groups itself
○
These lipids act as second messengers - the membrane is enriched with PIP2
generated by a kinase
○
This kinase phosphorylates on a hydroxyl group on the nocetol and generate PIP2
○
The kinase is activated by neuronal activity (exytosis)
○
So whenever there is exocytosis, there is more PIP 2 in the membrane and is
needed for the binding of AP2 to the plasma membrane
○
AP2 has a binding site for PIP2, same is true for AP180.
○
○
Dynamin also has a receptor for PIP2
○
However, in order for the un-coating of vesicles is inhibited by PIP2, so to
be able to un-coat the vesicles, PIP2 must peace out
You don't want the un-coating of membranes that haven't been
endocytosed yet
○
Only then do you want the dissembing of the clathrin lattice
○
○
PIP2 can be phosphorylated in order to go away
○
Endocytosis is basically completely regulated by PIP2
○
How many synaptic vesicles make it back to the active zone?
○
Is there some active transport mechanism used to bring vesicles back to the
active zone?
○
Is there a mechanism that synaptic vesicles make it back to the active zone
rather than some other part of the plasma membrane?
The neurotransmitter if released somewhere else it would not elicit a
response
○
○
Is the process bringing them back to the active zone regulated?
○
Actin may facilitate transport, acts as a railway track for synaptic vesicles from their
point of endocytosis back to the pool and eventually to the active zone
○
Diffusion of synaptic vesicles may be sufficient to account for this transport
○
Actin may also prevent the vesicles from reaching the plasma membrane
Because vesicles are bound to actin, they may not make it to the membrane
○
Might be. Not too sure if this is actually the case :-3 YAY science
○
○
Synapsin is a protein that associates with synaptic vesicles (green things in the
photo)
They bind to the vesicle membrane as well as actin
○
This connection is modulated by phosphorylation
○
○
Mechanism that keeps synaptic vesicles from getting to the synaptic zone
If you knock out synapsin genes, you get synapses that fatigue much more
quickly
○
Synapsin may be involved in creating a reserved pool of synaptic vesicles
○
○
How do vesicles know they need to return to the active zone? - may be the
role of Rab3 which is a small G protein which means that it binds GTP and
will then have a high affinity for the synaptic vesicle membrane
○
GTP has a partner called RIM
Once the synaptic vesicle has RAB3, it will make it more likely to
interact with the protein complex across the post synaptic neuron
○
Once fusion has occurred, RAB3 will dissociate from the membrane
○
RAB 3 is like a zipcode protein
○
○
Lines the plasma membrane at the synapse which allows vesicle docking
○
Pre synaptic specialization can be structurally variable
You can use a special technique called EM tomography which allows you
to tilt your specimen and visualize it from different angles and therefore
visualize it in 3D . If you do this with multiple sections, you are able to
reconstruct it
○
The neuromuscular junction contains two lines of synaptic vesicles that
are bordering the cytoplasmic active zone
○
○
Units of active zone on the cytomatrix
What are the proteins that make up this structure?
○
○
Allows for priming and docking at the plasma membrane for synaptic vesicles
○
Allows for calcium channels to be highly mobilized
Voltage gated calcium channels must be very close to the vesicles in order
to elicit release
○
Requires the recruitment of calcium channels next to this area
○
○
Allow for trans synaptic contact between two neurons
Make sure that there is a post synaptic cell fusion molecules
○
○
Munc13 will bind to RAB 3 and RIM
It is involved in priming - allows for close interaction between interaction between
RIM and RAB3
○
Will also bind to syntaxin and help unfold it in order for it to interact with
synaptogravin and SNAP 25
○
It contains a number of protein domains that allow for the regulation of its
functions
○
Can bind to calcium, it has a C2 domain and we know that when it binds calcium,
it can easilly unbind syntaxin
○
○
It also binds to lipid second messengers such as dicylglyercol
Anything signals and activates a metobotropic receptor will create dicylglycerol
which can bind to Munc13 which will be able to bind to syntaxin and SNAP 25
and therefore be able to unbind the SNARE complex
○
○
Will bind to modulat which binds to calcium and therefore accounts for facilitation of
priming received with calcium
○
RIM is a protein that interacts with Rab3 which is required to bring vesicles to
the active zone cytomatrix
○
RIM also interacts with calcium channels, it has been shown that RIM is
necessary to recruit calcium channels to the active zone cytomatrix
○
Allows for very quick vesicle fusion when calcium enters the presynaptic
terminal via voltage gated calcium channels
○
Connection to the post synaptic neuron
Synaptic Cell Adhesion Molecules
Neurexins and their interaction proteins and the Protein Tyrosine
Phosphatase are the two classes
○
All of them are recruited to the active zone cytomatrix by
scaffolding neurons such as ASK which are embedded into the
active zone cytomatrix because these alpha lipids bind to other
scaffolding proteins such as RIM
○
This complex allows for post synaptic specialization
○
○
○
Neurotransmitter Release 2
October(26,(2015
1:57(PM
Exocytosis
Overview: Transmitter exocytosis requires priming and calcium-dependent fusion of synaptic vesicles
○
Proteins involved in synaptic vesicle fusion with the plasma membrane
The SNARE complex brings synaptic vesicle membrane and plasma membrane into close
proximity
○
Clostridial neurotoxins inhibit transmitter release by cleaving SNAREs
○
Potential role of the SNARE complex in membrane fusion
○
Munc18/nSec1 may regulate SNARE complex assembly and facilitate membrane fusion
○
NSF and Ĵ-SNAP are involved in the disassembly of SNARE complexes
○
Summary: The cycle of SNARE complex assembly and disassembly The calcium sensor for
synaptic vesicle fusion
○
Synaptotagmin I is a calcium- and phospholipid-binding protein
○
Evidence that synaptotagmin is the calcium sensor responsible for calcium-dependent fast
transmitter release.
○
Hypothetical mechanism of synaptotagmin action
○
○
Firstly, synaptic vesicles need to make it to the plasma membrane (this is called synaptic vesicle
docking)
○
Secondly, the vesicles have to be tethered to the plasma membrane - there are protiens that are specially
required to do this. This is called synaptic vesicle priming, only then can the vesicles fuse with the
membrane once calcium rises from the pre synaptic specialization
○
Transmitter Exocytosis Requires Priming and Calcium-Dependent Fusion of Synaptic Vesicles
During priming, the synaptic vesicle membrane and plasma membrane are brought into close proximity
through interactions of synaptic vesicle membrane proteins and plasma membrane proteins.
They have to make it to the synaptic vesicle and tethered to the plasma membrane which is called
synaptic vesicle priming
○
Priming requires 3 or 4 proteins called SNARE proteins (Snap Receptors)
○
○
Cooperative calcium binding to the calcium sensor leads to fusion of the closely apposed membranes
○
The SNARE Complex Brings Synaptic Vesicle Membrane and Plasma Membrane Into Close Proximity
Membrane fusion generally involves SNAREs (SNAP receptors): Proteins with 70-aa residue SNARE
motifs.
They are proteins that have long alpha helical structures - they are protein structures that look like
springs. They are coiled and they have 3 or 4 SNARES
○
There are fatty acids that help it stick to the plasma membrane
○
These alpha helices like to form a bundle - they are energetically favourable so it is hard to pull
them apart again
○
These two membranes are negatively charged (phospholipids) the two membranes will then repel
each other - the energy required for this is taken from the formation of the SNARE complex
○
○
Three or four SNARE proteins on the two fusing membranes form a SNARE complex: Assembly of
SNARE motifs into a parallel four-helical bundle.
○
Synaptic vesicle exocytosis is mediated SNAREs syntaxin and SNAP-25 (plasma membrane), and
synaptobrevin (SV membrane).
○
The SNARE complex is assembled by formation of a helical bundle of one SNARE motif of each
synaptobrevin and syntaxin and two SNARE motifs of SNAP-25.
○
Synaptobrevin resides in the synaptic vesicle membrane
○
Syntaxin resides in the plasma membrane
○
SNAP 25 is associated with the plasma membrane, there are fatty acids attached to the peptide which
allows it to stick to the plasma membrane
○
These three proteins together can form a complex
Alphahelicies can form coils and are energetically favourable which makes it very hard to pull
them away from each other
○
They are negatively charged so the two membranes repel each other, the force that pushes them
together is generated from the formation of the SNARE complex
○
○
Clostridal Neurotoxins Inhibit Neurotransmitter Release By Cleaving SNARE Proteins
Clostridial neurotoxins responsible for tetanus and botulism are highly specific proteases that block
neurotransmitter release by cleaving SNAREs
○
Inhibition of neurotransmitter release by botulinum toxins and tetanus toxin shows that SNARE
complex formation is required for synaptic vesicle fusion
○
Bacteria that have toxins that paralyze neurons in the CNS - these toxins can be fatal
Tetanus and botulism are caused by this
○
However, if you inject these toxins, the neuromuscular junction between motor neurons in your
skin and the smooth muscle cells become paralyzed which gets rid of wrinkles
○
○
If these toxins are on the synapse, you get complete restriction of neurotransmitter release
○
They cleave the SNARE proteins and restrict release
○
Potential Role of the SNARE Complex in Membrane Fusion
Free energy gained during the formation of the SNARE complex (the four-helical bundle is
energetically far more stable than the unstructured SNARE motifs) may be utilized for the energetically
unfavorable membrane fusion.
○
SNAREs reconstituted in liposomes in vitro cause membrane mixing in the absence of any other
proteins: SNARE complex may be "minimal machinery" sufficient for membrane fusion.
○
SNARE complex, however, is not sufficient for kinetically efficient membrane fusion. Other proteins
are required to keep SNARE complexes in a position favorable for membrane fusion (a), or to
structurally organize several SNARE complexes that have to act cooperatively (b).
○
Munc18/nSec1 May Regulate SNARE Assembly and Facilitate Membrane Fusion
Munc18-1 (Ł nSeq1), is essential for neurotransmitter release: Genetic ablation causes complete
inhibition of both calcium-dependent and spontaneous neurotransmitter release.
○
SM proteins - Munc18 is a member of this group (Sec1/Munc18) proteins participate in all intracellular
membrane fusion reactions.
○
Two function have been proposed for Munc18-1:
Munc18-1 binds to syntaxin (protein in the plasma membrane) in its "closed" conformation and
(N-terminal domain folded back on the SNARE motif, inhibiting SNARE complex formation):
Munc18-1 may regulate syntaxin recruitment into SNARE complexes
○
Munc18-1 also binds tightly to syntaxin in SNARE complexes. Munc18-1 greatly enhances
fusion of synaptobrevin-containing liposomes with syntaxin and SNAP-25-containing liposomes:
Munc18-1 may be an essential part of the fusion machinery itself, facilitating SNARE
complexmediated fusion.
○
Munc 18 is necessary to form SNARE complexes on one synaptic vesicles - without it, it will
abolish all release
○
○
Lecture Notes:
SM proteins, in which Munc18 is a member of, has a dual function
They bind to syntaxin (which remember is in SNARE protein in the plasma membrane) and
then binds to SNAP 25 and synaptogravin but it can only do so if there is a conformational
change. In it's closed form, it folds back on itself and is unable to interact with SNAP 25 or
synaptogravin and it needs to be unfolded. Munc18, binds to syntaxin in its closed form, so
they believe it has a role in unfolding the complex
○
Munc18 can also bind to already assembled SNARE complexes, it may be necessary to
coordinate the coordination of several SNARE complexes in the vesicle
○
○
○
NSF and Ĵ-SNAP are involved in the disassembly of SNARE complexes
SNARE complexes need to be disassembled after fusion to allow reuse of the individual SNAREs
○
SNARE complexes, however, are extraordinarily stable. A chaparone (protein folding enzyme) is
needed to dissociate it
○
NSF is the cognate chaperone for SNARE complexes. It binds to the complex via an adapter protein, Ĵ-
SNAP. NSF then unwinds the Ĵ-helical bundle of the SNARE complex, using ATP as an energy source
○
SNARE is needed to bring the two membranes together and it is due to these two proteins it is hard to
bring back together again
Chaperon proteins fold and unfold proteins - it allows for formational changes using ATP or GTP
○
Chaperon proteins often come with adapter proteins, SNAP is exactly that.
SNAP binds to the SNARE complex
○
○
It then recruits NSF which unfold the complex - this allows us to reuse the three proteins
(Syntaxin, SNAP 25 and Synaptogravin)
○
○
The cycle of SNARE complex assembly and disassembly
Current model of SNARE complex assembly and disassembly:
○
Synaptotagmin I is a calcium- and phospholipid-binding protein
Synaptotagmin 1 or 2, they are associated with different genes, is an abundant synaptic vesicle
membrane protein composed of a short N-terminal intravesicular sequence, a single transmembrane
region, and two cytoplasmic C2 domains.
The domain extends into the cytoplasm
○
C2 domains are proteins that bind calcium and phospholipids which are an imperative aspect of
membranes
○
Calcium is positively charged, so the more positive charges you have on Synaptotagmin, the
higher the affinity for this protein
○
It also binds to the SNARE complex
○
○
Both C2 domains of synaptotagmin 1 bind calcium: The C2A domain has binding sites for 3 calcium
ions, the C2B domain 2 sites. Calcium binding is cooperative.
They interact with amino acids and bind calcium
○
○
Synaptotagmin C2 domains also bind to phospholipid membranes in a calcium-dependent and calcium
in a phospholipid-dependent manner. The affinity of synaptotagmin for calcium is low in the absence of
phospholipids and increases to 5-10 µM in their presence.
○
Synaptotagmin also binds to SNARE complexes in a partly calcium dependent manner
It does its function at the SNARE complex
○
○
Evidence that syanptotamin is the calcium sensor responsible for calcium dependent fast transmitter
release
In synaptotagmin I knockout mast fast neurotransmitter release is absent - because the synchronous
release uses a different calcium sensor
○
In contrast, spontaneous, action potential- and calcium-independent release is unchanged, showing that
the synaptic vesicle fusion mechanism itself is not affected.
○
Also, asynchronous transmitter release (red arrows in figure) is unaffected in synaptotagmin-/- mice.
Asynchronous release is caused by synaptic vesicle fusions that are triggered in a calcium-dependent
manner, but with some delay, and has to rely on a different calcium sensor than synaptotagmin.
It uses a different calcium sensory in central neurons and occurs miliseconds after the action
potential
○
○
Post synaptic currents occur after the stimulation which is called asynchronous release which occurs
immediately (1-3 ms) after the action potential while synchronous release occurs hundreds of
miliseconds after the action potential
○
Hypothetical Mechanism of Synaptotagmin Function
Synaptotagmin binds to the SNARE complex via a polybasic region in the C2B domain before Ca2+
influx
○
Calcium binding leads to additional electrostatic charges on the C2A and C2B domains, causing them
to bind negatively charged phospholipid membranes. The C2B domain has been shown to bind to two
different phospholipid bilayers, such that it likely aids in bringing synaptic vesicle membrane and
plasma membrane into very close apposition.
○
The close apposition of two negatively charged phospholipid bilayers is energetically unfavorable. This
promotes membrane fusion
○
When you have calcium influx, the domains (both C2A and C2B) gets positively charged which attracts
negative charges
Because both membranes are negatively charged (energetically unfavourable), the layers fuse
with each other to make a fusion pore
○
○
A kind of endocytosis that requires the construction
of a proteins lattice over the protein that needs to be
endocytosed. This is called nucleation.
The Lattice that is necessary for endocytosis needs to
be dissembled again.
For each synaptic vesicle that is exocytosed, you need competitor
endocytosis
So one vesicle will get endocytosed
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Endocytosis occurs according to a mechanism called clatherin mediated
endocytosis which is a type of endocytosis that requires the formation of a
protein lattice over the membrane that will be endocytosed
This is called nucleation
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Once the lattice is formed, the membrane graduates until you have a
vesicle stock. After the stock is formed, a protein complex arrives and
form around the stock and pinches off at the vesicle stock and can create a
new vesicle
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Clathrin needs adapter proteins, one being AP-2 which binds to
synaptogravin. Once it has bound to proteins in the synaptic membrane, it
recruits another protein called AP180 which recruits clathrin
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Nucleation requires the formation of this lattice made up of clatherin
Clatherin is made up of two proteins, a clatherin light chain
and a clatherin heavy chain
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Two clatherin heavy chains will interact with three light
chains and polymerize
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Dynamin pinches off the synaptic vesicles after polymerizing around
the stock (see photo). Actin is very important in the fission process
by helping dynamin pinch off the synaptic vesicle. Actin exists in
two forms, G Actin and F Actin. F actin makes long filaments
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Adapter proteins allow clatherin to bind to the plasma membrane
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AP-2 binds to many proteins that need to be endocytosed
Once it binds, it will recruit another adapter protein called AP180
Clatherin nucleation cannot occur without the binding of
AP180 to AP2
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Hsc70 is another adaptor protein that assembles the AP-2 and AP180
complex for clathrin. It needs auxilin which is an adaptor protein to make
the lattice.
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Dynamin can polymermarize and will pinch off the synaptic vesicles from the stock
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Can't have any more endocytosis than exocytosis or else your plasma membrane will shrink, so
they must be tightly linked. One concept is the regulation of phsophatidylinositol which are
lipids that have a sugar that can have phosphate groups themselves. The lipids act as second
messengers.
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Actin is important in the fission process by helping dynamin to pinch off the vesicles
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Actin exists as G Actin and F actin, G actin is a monomer while F actin is a polymer
F actin makes very long filaments
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Hsc70 is also a chaporon protein and dissasembles the AP-2 and AP180 complex
It needs auxilin to bind to the clatherin lattice
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Endocytosis and exocytosis are tightly linked through phosphatidylinositides
They have fatty acids and glyercol, and a sugar called nocetol which can have
phosphate groups itself
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These lipids act as second messengers - the membrane is enriched with PIP2
generated by a kinase
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This kinase phosphorylates on a hydroxyl group on the nocetol and generate PIP2
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The kinase is activated by neuronal activity (exytosis)
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So whenever there is exocytosis, there is more PIP 2 in the membrane and is
needed for the binding of AP2 to the plasma membrane
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AP2 has a binding site for PIP2, same is true for AP180.
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Dynamin also has a receptor for PIP2
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However, in order for the un-coating of vesicles is inhibited by PIP2, so to
be able to un-coat the vesicles, PIP2 must peace out
You don't want the un-coating of membranes that haven't been
endocytosed yet
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Only then do you want the dissembing of the clathrin lattice
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PIP2 can be phosphorylated in order to go away
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Endocytosis is basically completely regulated by PIP2
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How many synaptic vesicles make it back to the active zone?
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Is there some active transport mechanism used to bring vesicles back to the
active zone?
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Is there a mechanism that synaptic vesicles make it back to the active zone
rather than some other part of the plasma membrane?
The neurotransmitter if released somewhere else it would not elicit a
response
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Is the process bringing them back to the active zone regulated?
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Actin may facilitate transport, acts as a railway track for synaptic vesicles from their
point of endocytosis back to the pool and eventually to the active zone
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Diffusion of synaptic vesicles may be sufficient to account for this transport
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Actin may also prevent the vesicles from reaching the plasma membrane
Because vesicles are bound to actin, they may not make it to the membrane
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Might be. Not too sure if this is actually the case :-3 YAY science
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Synapsin is a protein that associates with synaptic vesicles (green things in the
photo)
They bind to the vesicle membrane as well as actin
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This connection is modulated by phosphorylation
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Mechanism that keeps synaptic vesicles from getting to the synaptic zone
If you knock out synapsin genes, you get synapses that fatigue much more
quickly
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Synapsin may be involved in creating a reserved pool of synaptic vesicles
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How do vesicles know they need to return to the active zone? - may be the
role of Rab3 which is a small G protein which means that it binds GTP and
will then have a high affinity for the synaptic vesicle membrane
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GTP has a partner called RIM
Once the synaptic vesicle has RAB3, it will make it more likely to
interact with the protein complex across the post synaptic neuron
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Once fusion has occurred, RAB3 will dissociate from the membrane
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RAB 3 is like a zipcode protein
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Lines the plasma membrane at the synapse which allows vesicle docking
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Pre synaptic specialization can be structurally variable
You can use a special technique called EM tomography which allows you
to tilt your specimen and visualize it from different angles and therefore
visualize it in 3D . If you do this with multiple sections, you are able to
reconstruct it
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The neuromuscular junction contains two lines of synaptic vesicles that
are bordering the cytoplasmic active zone
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Units of active zone on the cytomatrix
What are the proteins that make up this structure?
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Allows for priming and docking at the plasma membrane for synaptic vesicles
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Allows for calcium channels to be highly mobilized
Voltage gated calcium channels must be very close to the vesicles in order
to elicit release
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Requires the recruitment of calcium channels next to this area
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Allow for trans synaptic contact between two neurons
Make sure that there is a post synaptic cell fusion molecules
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Munc13 will bind to RAB 3 and RIM
It is involved in priming - allows for close interaction between interaction between
RIM and RAB3
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Will also bind to syntaxin and help unfold it in order for it to interact with
synaptogravin and SNAP 25
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It contains a number of protein domains that allow for the regulation of its
functions
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Can bind to calcium, it has a C2 domain and we know that when it binds calcium,
it can easilly unbind syntaxin
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It also binds to lipid second messengers such as dicylglyercol
Anything signals and activates a metobotropic receptor will create dicylglycerol
which can bind to Munc13 which will be able to bind to syntaxin and SNAP 25
and therefore be able to unbind the SNARE complex
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Will bind to modulat which binds to calcium and therefore accounts for facilitation of
priming received with calcium
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RIM is a protein that interacts with Rab3 which is required to bring vesicles to
the active zone cytomatrix
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RIM also interacts with calcium channels, it has been shown that RIM is
necessary to recruit calcium channels to the active zone cytomatrix
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Allows for very quick vesicle fusion when calcium enters the presynaptic
terminal via voltage gated calcium channels
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Connection to the post synaptic neuron
Synaptic Cell Adhesion Molecules
Neurexins and their interaction proteins and the Protein Tyrosine
Phosphatase are the two classes
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All of them are recruited to the active zone cytomatrix by
scaffolding neurons such as ASK which are embedded into the
active zone cytomatrix because these alpha lipids bind to other
scaffolding proteins such as RIM
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This complex allows for post synaptic specialization
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Neurotransmitter Release 2
October(26,(2015
1:57(PM
Document Summary
Overview: transmitter exocytosis requires priming and calcium-dependent fusion of synaptic vesicles. Proteins involved in synaptic vesicle fusion with the plasma membrane. The snare complex brings synaptic vesicle membrane and plasma membrane into close proximity. Clostridial neurotoxins inhibit transmitter release by cleaving snares. Potential role of the snare complex in membrane fusion. Munc18/nsec1 may regulate snare complex assembly and facilitate membrane fusion. Summary: the cycle of snare complex assembly and disassembly the calcium sensor for synaptic vesicle fusion. Evidence that synaptotagmin is the calcium sensor responsible for calcium-dependent fast transmitter release. Firstly, synaptic vesicles need to make it to the plasma membrane (this is called synaptic vesicle docking) Secondly, the vesicles have to be tethered to the plasma membrane - there are protiens that are specially required to do this. This is called synaptic vesicle priming, only then can the vesicles fuse with the membrane once calcium rises from the pre synaptic specialization.
