Likewise, a series of experiments were done that further this theme, which
illustrate that Calcium is both necessary and sufficient to trigger vesicle fusion
and the release of neurotransmitter. Now using the same preparation, it's
possible to inject Calcium directly into the presynaptic terminal.
And when you do that, you can measure a depolarization of the postsynaptic
neuron. So, this suggest that infusion of Calcium
to the presynaptic terminal leads to some vesicle fusion events, the release of
neurotransmitter, the opening of cation selective channels in the postsynaptic
membrane, and the depolarization of that cell.
Here, again, is further evidence for the importance of Calcium in the controlled
experiment. The presynaptic membrane is depolarized
such that an action potential is generated.
About a millisecond later, an action potential is recorded in the postsynaptic
cell. But if a Calcium chelator is injected
into the presynaptic cell, this is a compound that is going to prevent a
sudden rise of intercellular Calcium. So, so now we're not blocking the Calcium
channel, we're just sucking up the Calcium that enters the presynaptic
terminal. So, even though we have a nice robust
action potential In the presynaptic neuron, there's only a very modest
sub-threshold depolarization seen in the postsynaptic cell.
So, this experiment suggests that a sudden rise of Calcium in the presynaptic
terminal was critical for the release of neurotransmitter and the generation of an
action potential in the postsynaptic cell, Okay?
Well, so far, we haven't really discussed how all of this comes about.
And to begin that discussion, we need to consider some of the molecules that are
affiliated with the synaptic vesicle membrane, as well as the membrane at the
presynaptic terminal. So, here's a rather fanciful look at a
synaptic vesicle. And what we see is an artist rendition,
now a couple of years old but still just conveys the point of the complexity from
a molecular perspective of the surface of the vesicular membrane.
And this complexity reflects the precise means by which the presynaptic terminal
regulates the trafficking of synaptic vesicles and controls their fusion with
the presynaptic terminal. So, I show you this just to make the
point that there are dozens of molecules in the vesicular membrane that are
important for regulating the activity at a chemical synapse.
One of these molecules is particularly important, and I will highlight that for
you. It's a protein called Synaptotagmin.
And here, it is illustrated here. So, Synaptotagmin appears to be the
molecule in the vesicular membrane that binds to Calcium.
So, as Calcium rushes into that presynaptic terminal, Synaptotagmin seems
to be the Calcium sensor that interacts with Calcium.
And eventually, sets into motion the events that quickly unfold leading to the
fusion of the membrane in the pre synaptic terminal.
The way that this happens is through the activation of the set of proteins, some
of which are found in the synaptic vesicle membrane.
Others are found in the membrane of the presynaptic plasma membrane.
Together, these proteins form something called the SNARE Complex.
So, SNARE is actually an acronym. you don't need to be concerned with
knowing that acronym but it's helpful to use this acronym because it reminds you
as to the function of this complex of proteins.
So, the SNARE complex is involved in snaring the vesicle membrane to the
cytoplasmic face of the presynaptic terminal.
This is, in fact, the docking process. So as these membranes approach one
another there is an interaction among these proteins.
Basically, these proteins, such as Synaptobrevin SNAP-25, and some others,
they associate with one another and perhaps form some sort of helical
interaction that allows the synaptic vesicle to be docked to the presynaptic
terminal. Now, notice the proximity of the SNARE
complex to Synaptotagmin. Synaptotagmin, again, is the molecule
that binds Calcium ions as Calcium enters this presynaptic terminal with the
arrival of depolarization. So, let's sort of step through that
process and see how this works. Initially, we have proteins affiliated
with the vesicle membrane, we have Synaptobrevin, SNAP-25 and Synaptotagmin.
But, not yet do we have sig, significant depolarization arriving.
So, the SNARE complex begins to dock a vesicle membrane.
And this happens in a Calcium independent fashion.
This allows a vesicle to be docked, even without an inrush yet of Calcium into the
presynaptic terminal. So, when the SNARE complex forms, we can
say that these membranes have been pulled together, and this vesicle is now docked.
So, this is a picture of what a docked vesicle looks like.
The SNARE complex is formed, this vesicle is primed and ready for action with the
arrival of a wave of depolarization. So, once that happens voltage-gated
Calcium channels open, Calcium rushes into the presynaptic terminal.
And now, Calcium binds to Synaptotagmin. And the interaction of Calcium in
Synaptotagmin leads to a series of confirmational changes in the SNARE
complex that tugs these two membranes together.
And as that happens, then the membranes will fuse and neurotransmitter now is
free to diffuse out of the synaptic vesicle through what's called Fusion Pore
that forms as the vesicle membrane in the presynaptic terminal unite.
Well, now that the neurotransmitter is diffused out of the synaptic vesicle
what's left is the retrieval of the membrane from the synaptic vesicle.
And this involves a variety of proteins some of which are illustrated here in
this slide. I would highlight just a couple of them
for you. one that's very important is this protein
called Clathrin. so Clathrin coats the vesicle membrane
and with interactions with other proteins such as this Dynamin protein.
we have the retrieval of those membranes, and here's a picture of what this looks
like. So, the Clathrin molecule has an
interesting geometrical shape. We see it up here to the upper left.
It forms a structure called a Triskelion, and that Triskelion then can associate
with other molecules and essentially form this buckyball-like ring around the
membrane. And so, as these Clathrin, Triskelion
begin to form, then the vesicle membrane is pulled back away from the presynaptic
terminal. at some point in here, Dynamin gets
involved and appears to nip these vesicle membranes, allowing for a completely
coated vesicle membrane to now be retrieved.
And via interactions with Actin filaments and a variety of other proteins this
vesicle membrane is retrieved. The Clathrin coat is removed, and the
vesicle is now ready for repackaging with neural transmitter without loss of all of
those specialized proteins that are associated with this vesicle.
Like Synaptotagmin in the SNARE complex that are important for the function of
this membrane as it's recycled and repackaged with more neurotransmitter.
Now, I'd like to conclude this tutorial by thinking about the activity of a toxin
that's found in nature, from the Clostridium family of bacteria.
And it's over the last couple of decades or so, has been used relatively widely in
clinical practice. and also, unfortunately been somewhat
abused perhaps, in the cosmetic industry. And, of course, what I am speaking of is
Botulinum Toxin, otherwise known as Botox.
So, Botox can be used in the therapeutic setting to reduce muscle tension, to
reduce muscle spasticity perhaps to quiet a twitching muscle.
these are all very good and appropriate uses of botox.
botox can also be used cosmetically to try to reduce muscular activity that
might give rise to wrinkles or furrows in the skin.
And for a variety of reasons, botox has become a fairly widely used drug in our
time. So, how does it work?
Well, the way botox works is by cleaving the SNARE complex.
So, botox is a Protease. And it is a Protease that disrupts the
SNARE complex in one of several sites depending upon exactly what formulation
of Botulinum Toxin we're speaking of. one of the most common and widely used
forms of botox is, is BoTX-A, and we now know exactly where.
The cleavage site for BoTX-A is within the SNARE complex.
And a variety of other botox formulations have their own cleavage sites on one
member or another of the SNARE complex. So, you can imagine why this works so
well. So, if the SNARE complex is cleaved, then
no matter how much Calcium rushes into the presynaptic terminal, that vesicle is
very unlikely to fuse on it's own. The SNARE complex is what facilitates
bringing together these membranes and twisting and turning, resulting in the
fusion of the vesicle membrane with the presynaptic terminal.
Without the SNARE complex to pull those membranes together, we're very unlikely
in having a fusion event. Therefore, we're very unlikely to release
a neurotransmitter at that synaptic terminal.
So, botox is a very powerful molecular tool that can be used to silence a
chemical synapse. Okay?
Well, we've come to the end of the tutorial.
And I would encourage you to view one of the animations that will review these
processes that are involved in synaptic transmission.
It's animation 5.1. You can follow the hyperlink at the end
of the handout or navigate there via our website.
Next time, we'll talk about neurotransmitters, and after that, the
receptors for neurotransmitters. So, I'll see you then.
