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Learning Objectives
Review background information required from previous taught material
Discuss the key steps in fast chemical synaptic transmission
Appreciate how chemical synaptic transmission impacts on membrane voltage
Neuronal Signalling
Information transfer in the brain – brain needs to integrate electrical and chemical signals
We concentrate on 1 & 2 as these are the forms of cellular communication that are specialised
and dominant in an adult system.
All cells have an innate permeability – they allow ions to flow across a plasma membrane that on
its own would be impermeable to ions.
This permeability is because of ion channels – specialised proteins found in the membranes of
virtually all cells that allow certain ions to flow.
In order for ions to flow, there needs to be a differential gradient of ions across the membrane to
begin with. Without this ions channels would not do anything – there would be no force driving the
flow of ions (ion flux).
The asymmetrical distribution across the membrane (difference in ionic strength) is due to the
presence of pumps.
Pump: An energy source (e.g. ATP) translocates ions across the membrane. Does not contain a
pore, the ions are physically moved across the membrane.
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Molecular Cell Biology II Neuronal Signalling
The pump builds up an ionic gradient. If appropriate ion channels were to open then ion flux would
occur.
Nernst equation
Variables: Constants:
[X] = concentration of ion (moles) R = gas constant (8.314 J K-1 mol-1)
z = charge or valence of transported X T = temperature (298 K)
F = faraday (96490 J V-1 mol-1
Given the external and internal concentrations of an ion, the Nernst equation can be used to
predict the driving force – at any given voltage, how much current will flow (due to the ionic
gradient).
If intracellular and extracellular concentrations of K + are plugged into the equation, then the
equilibrium potential for potassium ions (K+) will be estimated.
This means that if the membrane potential was at -100 mV, the system for K + is at equilibrium
This voltage will offset the ionic gradient
If a K+ channel were to open at this membrane potential there would be no
K+ flux
If the membrane were more depolarised (e.g. -70 mV) then there will be a driving force causing
K+ to flow.
This is down to the charged amino acids that line the pore of the channel. For a channel relating to
positively charged ions, the amino acids in the pore will have a negative charge, to attract the
positively charged ions.
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Molecular Cell Biology II Neuronal Signalling
The further the membrane voltage is from equilibrium potential, the greater the current flow
The RMP (resting membrane potential) of a cell is a steady state voltage that the cell is at most of
the time. The cell cannot reach any particular equilibrium completely.
RMP = -70 mV
The -100 mV equilibrium potential for K+ is never reached, due to the influence of other ions
Na+ equilibrium potential = +50 mV
Ca2+ can also have an effect
2. Depolarisation
Depolarisation moves the membrane potential to a threshold voltage
The threshold voltage is the voltage at which voltage-gated sodium channels are opened
Ion channels have gating parameters which define what will open/close them
In this case, the sodium channels are sensitive to the membrane voltage
The AA composition and structure of the channel is involved in the voltage sensitivity
These channels are thought to have a ball and chain method of inactivation, a loose part of the
protein can plug the channel.
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Molecular Cell Biology II Neuronal Signalling
Ion channels don’t stay open very long. They are inactivated soon after opening, despite
being at the right voltage. It is important not to have the channel open for a long time.
At ~ +40 mV the K+ channels will open, dragging the voltage back.
3.
Repolarisation
Na+ channels get inactivated
Permeability to K+ increases
4. Hyperpolarisation
The K+ channels remain open after repolarisation
The huge influx of K+ causes the membrane potential to become even more polarised than the
RMP. The membrane potential gets very close to the equilibrium potential for
K+.
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Molecular Cell Biology II Neuronal Signalling
The K+
channels are eventually closed and the system can return to its steady state – RMP
The period between 4. and the return to RMP is known as the AHP (after hyperpolarisation)
The membrane still has high K+ permeability
This can determine the refractory period of a system
During the AHP the cell cannot fire any more action potentials, it is insensitive
This can determine the firing patterns of neurons and their max frequency to fire
Some cells need to fire at high frequencies, and so have brief AHPs
o e.g. Interneurons in the Hippocampus
o Need to encode very fast temporal information in frequency range
Other cells do not require this, so have a longer AHP
o e.g. dopaminergic neurons, involved in Parkisons disease
o Broad, slow action potentials
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Molecular Cell Biology II Neuronal Signalling
Myelination:
Functions…
1. Insulation
The voltage gated channels that are surrounded by myelin cannot do anything
Even if they could open, the insulating myelin would prevent ion flow
The myelin creates a separation from the extracellular space
Action potential reaches the synaptic terminal, and needs to pass information onto the next neuron
across a gap via its dendrites.
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Molecular Cell Biology II Neuronal Signalling
Ca2+ is kept at low levels in all cells as it is a dangerous signalling molecule and needs to
be controlled – it can trigger cell death.
Neurons have buffering capacity for Ca2+ as they have many Ca2+ binding proteins
Very sensitive and fast – rapid detection
Change in membrane voltage causes a conformational change in the VGCCs
Voltage clamp technique allowed determination of time taken to open VGCCS after
electrical signal arrived (0.5 ms)
When the Ca2+ channels open there is a huge influx of calcium ions, raising the intracellular
concentration of calcium significantly, for a short period of time. Calcium binding proteins will ‘mop
up’ the ions quickly. The entry of calcium triggers exocytosis.
3. Exocytosis
Ca2+ causes vesicles containing a high concentration of neurotransmitter to fuse with the pre-
synaptic membrane, releasing their contents into the synaptic cleft
4. Diffusion
Neurotransmitter diffuses across the synaptic cleft
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Molecular Cell Biology II Neuronal Signalling
The black lines show single channel activity, they open/close at a high frequency
There is also a slight lag (0.5 ms) when the membrane potential goes back to -70 mV, the
channels do not close instantly.
The greatest influx of calcium occurs just before the channels are
closed, this is known as the tail current.
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Molecular Cell Biology II Neuronal Signalling
The synaptic cleft is the region between the post and pre-synaptic membranes
It is very narrow ~50 nm
Neurotransmitters will diffuse across this space rapidly
Neurotransmitter will then bind to receptors on the post-synaptic membrane
The receptors are ligand gated ion channels
Vesicle Fusion:
It is thought that full fusion does not occur, a “kiss-and-run” phenomena dominates instead
Synaptic Delay:
There is a significant delay between the influx of calcium and the postsynaptic response (200 µs)
The relationship between Ca2+ concentration and neurotransmitter release is not linear, results
show that the trend is more exponential (raised to the power of 4). This makes the system very
sensitive to calcium and the 4th power relationship might be significant in terms of the calcium
sensor itself.
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Molecular Cell Biology II Neuronal Signalling
6. Binding to receptors
Neurotransmitter molecules bind to specialised receptors (ligand-gated ion channels) in the
postsynaptic membrane. The ligand is the neurotransmitter.
Can also bind to G-protein coupled receptors (GPCRs), which do not have intrinsic activity on ion
channels, however do have an action. Slower time for a response (seconds – minutes).
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Molecular Cell Biology II Neuronal Signalling
2. ACh diffuses rapidly, then binds to the ACh receptors (AChR) found on the muscle
ACh receptors also known as nicotinic receptors as nicotine binds strongly
Nicotine will cause desensitisation, causing receptor close for a long time
Synapses in the brain do work in a similar way to this however less is known about them
*In the peripheral nervous system of mammals, ACh is used instead of glutamate as the excitatory
transmitter
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Molecular Cell Biology II Neuronal Signalling
disrupted. AChR is the best-studied ligand gated ion channel. To overcome these issues, the
receptors were loaded into a micelle (an artificial liposome), ACh was added, then they were
quickly frozen.
The AChR has to respond very rapidly, making a fast conformational change
Once ACh binds to the two binding pockets (found on ), there is a rotation in the extracellular
domain. This makes it more energetically favourable for the protein to exist in a slightly different
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Molecular Cell Biology II Neuronal Signalling
conformational state. A rotation occurs in the pore, in the TM2 region. The channel size itself does
not change much, the charge within the TM2 region is exposed.
Two molecules of neurotransmitter have to bind for this process to occur. After the first ACh
binds, the affinity for binding the second increases (co-operativity).
The patch-clamp method has been used to study the ACh receptors in detail. A small region of
membrane is selected, then it is very briefly exposed to ACh. 1mM 1ms.
It showed that the channel do not like to be open (P open is low)
As with the voltage gated channels, they do not close immediately after the signal is taken away.
In this case it is due to the receptor having a very high affinity for the neurotransmitter. ~10 ms.
While neurotransmitter is bound, the channels open/close rapidly, they are not kept open.
This area of study has implications in many diseases affecting the CNS
* Synapse number / neuron can vary from 30 (cerebellar granule) – 300,000 (Purkinje)
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Molecular Cell Biology II Neuronal Signalling
Popen is the most important parameter (<0.3 usually, a faulty biological machine)
is a set, unchangeable parameter, determined by AA charge in the pore
Nc varies per synapse
An excitatory synaptic conductance (gs) results in a change in voltage (EPSP) that brings the
neuron closer to the action potential threshold.
tm= Rm.Cm
Rm = membrane resistance – reflects the resting permeability to Na +, K+, Cl- ion flux
Cm = membrane capacitance – reflects the total membrane area
Structure of neuron determines Rm and Cm and so is important for duration of voltage
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