Neuropharmacology is a very important field of neuroscience.
It forms the basis of the pharmaceutical industry
and our hopes for treating brain diseases.
There are many compounds that act specifically
at different receptors in the brain.
So for AMPA receptors we have CNQX and MBQX
for NMDA receptors we have APV and MK-801,
and for the GABA-A receptor we have antagonists
like picrotoxin, bicuculline and gabazine.
These antagonists have highly specific actions
and are extremely useful in the experimental setting,
but therapeutically, they've not proven to be useful.
In this video we're going to look at benzodiazepines
that have turned out to have interesting therapeutic potential
and have been extensively used.
Benzodiazepines were first developed in the '60s, and were marketed as Valium,
also known as diazepam, and since then many benzodiazepines
have been developed with higher specificities
and better therepeutic outcome.
It's interesting to look at the benzodiazepines,
because they have an extremely specific mechanism of action and a lot
is known about how they function at the molecular level.
That's what we'll explore in this video.
Let's begin by taking a closer look at the GABA(A) receptor.
The GABA(A) receptor is a pentameric structure,
made out of five different subunits,
each of which is an individual protein that comes together
to form the ligand-gated GABA(A) receptor with the chloride channel down the middle.
Each subunit is encoded by a single protein and there are
a number of different genes that code for those subunits.
They can be divided into different families.
There's an alpha, beta, gamma, rho, delta, pi, epsilon, theta —
types of GABA(A) receptor genes,
and in general for most GABA(A) receptors,
they're composed of two alpha subunits, two beta subunits and a gamma subunit.
The gamma subunit can also be one of these rho
or other ones in grey.
The GABA binding site turns out to be somewhere
between the alpha and the beta subunit.
So in fact, there are two GABA binding sites
between each of the alpha and beta subunits.
The benzodiazepine binding site, however, is at a different location
and sits between the alpha and the gamma subunits.
In fact, you need to have a gamma-2 or a gamma-3 subunit present
in the GABA receptor in order for the benzodiazepines to bind.
In terms of the amino acid structure of the GABA(A) receptor,
they form a large N-terminal region
where the GABA binding site is encoded
and also there's a benzodiazepine binding site.
We have four transmembrane alpha helices —
one, two, three and four —
and this one here turns out to be the [paw]-forming region
which then hosts the chloride ion channel.
The GABA and the benzodiazepine binding sites are sitting here
on the N-terminal which is on the outside of the cell.
The benzodiazepenes don't in themselves activate the GABA-A receptors,
so they bind to the GABA-A receptor but it's at a different site
where GABA activates the ion channel.
What the benzodiazepines do is that they increase the affinity
of the GABA-A receptor for GABA.
So GABA binds more tightly to it in the presence of benzodiazepines.
If we think about the electrophysiology, functionally relevant
in terms of the IPSCs mediated by synaptic transmissions —
that's inhibitory postsynaptic currents —
then we'll see that under control conditions
the IPSC has a certain amplitude and a certain duration.
If you then apply benzodiazepine, that then doesn't change the amplitude
of the IPSC, but it prolongs the duration of the IPSC.
So there's a total charge transfer that's mediated in the presence
of benzodiazepines that's larger,
and so it potentiates the GABA-A currents
and potentiates the inhibition in the brain.
So benzodiazepines have an interesting action.
They don't in themselves cause inhibition, but they only potentiate
the effect of GABA when it's normally released in the brain.
So they only act when they GABA's been released
at the right time and the right place in the brain.
So it's a highly specific potentiation of GABAergic inhibition
that the benzodiazepines mediate.
There's further specificity as to which type of GABA-A receptors
the benzodiazepines act upon.
It turns out that GABA-A receptors need to contain
alpha-1, -2, -3, or alpha-5 subunits
and if instead they have an alpha-4 or alpha-6 subunit,
then the benzodiazepines don't have any effect.
Now as the molecular biologist were beginning to clone and sequence
the different GABA receptor alpha subunits,
they found that there was in general very high homology,
so that the amino acid sequences of the different alpha subunits
were very similar, but there were of course also many differences.
Here in one particular sequence, alignment of the different subunits,
we see that the four that bind benzodiazpines
have a histidine at this position —
101 for the alpha-1 and alpha-2 subunits,
so these are the numbers counting from the N-terminal,
the amino acids, so we have the different amino acids here,
and at position 101 in both alpha-1 and alpha-2
we have a histidine.
Alpha-4 and alpha-6 in this equivalent position
have an arginine that's present.
So there's a difference in the amino acid structure
of the receptors that bind benzodiazepines and the ones that don't.
There are of course other differences in the amino acid sequences
of the GABA-A receptors, but you can at least test
to see whether this makes the difference for benzodiazepine binding.
So we can take the alpha-1 or the alpha-2 subunits
and change this histidine to an arginine
and now see: Does that make a difference to the benzodiazepine effect?
The molecular biologists have figured out
how to make changes to the amino acid sequence
of GABA-A receptors and they've also figured out
how to express them in heterologous systems
and so here we're expressing wild type GABA, alpha-1 subunit
together with a beta and a gamma subunit,
express them, apply GABA, and we see
a nice GABA-mediated chloride current.
In the presence of diazepam — that's the benzodiazepine Valium —
we apply that at one micromolar and now when GABA is applied
the GABA-mediated currents are much larger.
That's the potentiating effect of benzodiazpines upon the GABA-A receptor.
Now, in the mutants where we take the histidine
at position 101 and change it to an arginine —
that's what's typically present in the alpha-4 and alpha-6 subunits —
these are the ones that are insensitive to diazepam —
now we change just this one amino acid in the alpha-1 subunit
and now when you apply diazepam there's no potentiation
of the GABAergic currents.
It's the same in the presence of diazepam as it is without diazepam
when this point mutation has been carried out.
So it turns out that this histidine in position 101 is essential
for the diazepam, in order to mediate its effect
at potentiating the GABA-A receptor.
Interestingly, this point mutation makes no difference
to the currents evoked by GABA in the absence of diazepam.
So in many respects, this point-mutated GABA alpha-1 receptor
is identical to the wild type,
except in the presence of the exogenous agent diazepam
or other benzodiazepines.
This then allows us to do very interesting genetic testing
to see which subunits mediate which actions
of the benzodiazepines.
So in the mouse genome, we can also go and make
exactly the same mutations to the GABA-A receptors.
We can change — in the alpha-1 or the alpha-2 subunits —
we can take that histidine at position 101
and change it to an arginine and then we'll make GABA receptors
for these specific subunits that are insensitive to diazepam.
What's good about doing this at the genome level in mice
is that we can then see: What are the behavioral effects
of applying the drug — the benzodiazepine —
to an animal, in terms of the behavioral change
that diazepam makes.
We're interested in two different effects of the benzodiazepines.
One is the sedative action, and that's what we'll look at first.
If you take a wild type mouse and you put it inside an arena,
you can then track the position of that mouse as it runs around.
You can see where it goes
and you can quantify the distance that it moves.
If you then give that same mouse some diazepam
at a relatively high concentration — 30 mg/kg — that has
a sedative action on the mouse.
The mouse might go to sleep, or it will just relax
and do nothing, perhaps staying in this corner of the cage.
If you now take a different mouse where you've made the genetic mutation
so that the alpha-1 subunit is no longer sensitive to diazepam,
then the basal condition,
where there's no benzodiazepines present, the animal is just like wild type,
the GABA alpha-1 subunit behaves
just like the normal alpha-1 subunit
in the absence of diazepam, and so the mouse runs around,
explores its environment.
You give it the large dose of diazepam and in the point-mutant mouse
for the alpha-1 subunit, it has no effect.
The sedative effect of diazepam is gone.
So apparently, sedation is mediated by the alpha-1 subunit
of the GABA-A receptor.
You can test the specificity for it acting on the alpha-1 subunit
by doing the same mutation in the alpha-2 subunit.
So now we take a point-mutant mouse,
it has the histidine replaced by an arginine in position 101
for the alpha-2 GABA-A receptor, we give the diazepam,
and the sedative action still works.
So sedation doesn't work via the alpha-2 subunit,
but it's specifically mediated by the alpha-1 subunit
of the GABA-A receptor — a remarkable specificity.
The other interesting action of the benzodiazepines
is anxiolysis, a reduction in anxiety.
This we can also monitor in mice by thinking about their overall behavior
and what they're afraid of.
Mice are worried about large, open, light spaces.
They're nocturnal animals and they prefer to move around in the dark.
So experimentally in the lab, we can design behavioral arenas
that have a dark area and a light area.
If you put the mouse in this arena,
it will tend to run around in the dark area.
Every now and then, it might poke into the light area,
but it's afraid, it's anxious, and it will rapidly return to the dark area.
If you give these mice a small dose of diazepam, then they'll relax,
and their fears will be reduced; they're less anxious.
They'll then go and explore the light area
to a much greater extent than under the control conditions.
That's the anxiolytic effect of diazepam,
here occurring at much lower doses than the high concentrations for sedation.
We can then do the same two point mutations as before
and study the effects of diazepam.
In the alpha-1 point mutation, the diazepam still works.
It has the anxiolytic effect, and the mouse will explore the light area.
But in the alpha-2 point mutation
the diazepam fails to have the anxiolytic effect.
The point mutation here means that the diazepam can't act
upon the alpha-2 subunit and the fact that there's then
no anxiolytic relief from diazepam means that anxiolysis
is being mediated by the alpha-2 receptor,
at least in mice.
So we've seen that benzodiazepines act in a highly specific way
upon some some subtypes of GABA-A receptors.
It turns out that the sedative action of benzodiazepines
is mediated by the alpha-1 receptor and the anxiolytic effect
of benzodiazepines is activated via the alpha-2 receptor.
That, of course, is an analysis that's been done in mice,
and it's unclear to what extent that will carry over into the human situation,
but it has of course inspired the pharmaceutical industry
to go and make agents that are specifically acting
at alpha-1 and alpha-2 subunits, and of course, it's probably also
interesting to explore the other subunits of the GABA receptors that may have
different therapeutic actions.