Learning Objectives * Review

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

1. Action potentials – rely on neurons being highly polarised
* All cells do have an electrical potential, but it is less significant than in neurons
2. Synaptic transmission – enables the electrical signal to ‘jump’ between cells
* Intercellular communication
3. Gap junctions – allow passive transmission of an electrical signal to nearby connected cells
* Prevalent early on in development, rarely found in adults
* Intercellular communication

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.

In neurons the pump is a 2K+/3Na+ pump, with ATP being hydrolysed
* High [K+] inside the cell
* High [Na+] outside the cell
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.

For K+Veq = -100 mV

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.

The amount of current that flows is equal to a parameter known as ?
? is the conductance (a measure of permeability – the ability of an ion to move across membrane)
? permeability of channel = ? conductance (?)

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.

? negative charge in / around the pore = ? conductance

Current flow (I) = ?? (VM – Veq)

The further the membrane voltage is from equilibrium potential, the greater the current flow

During an action potential, all of these parameters will change
A neuron is never at equilibrium, it is undergoing constant changes

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

The Action Potential:

1. Resting membrane potential
The RMP is set by the resting permeability of the membrane to Na+, K+ and Cl- ions

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.

a) These channels will not be open at the RMP (-70 mV)
b) Once the membrane has been depolarised to around -50 mV, they start to open
* Membrane is now very permeable to sodium ions
c) Membrane rapidly moves towards +50 mV, trying to reach the equilibrium potential of sodium ions, but doesn’t reach this value…
* 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+.

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

* Not all neurons are myelinated
* e.g. peripheral nervous system – most neurons are not myelinated
* Myelination is not just down to Schwann cells
* e.g. in the brain it is due to oligodendrocytes
* Electricity will always take the path of least resistance
* Lipid bilayer is a capacitor (it stores charge)

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

2. Reduced capacity of load
* How does the cell ‘know’ which way to fire the action potential?
* This is an issue in long cells where the path of least resistance would be up the dendrite rather than down the long axon which has a much greater membrane area
* This large capacitance would force the AP into the dendrites, the wrong way
* Myelin reduces the capacitance
* Not an issue in unmyelinated neurons as they are short
Synaptic Transmission – Presynaptic:

Action potential reaches the synaptic terminal, and needs to pass information onto the next neuron across a gap via its dendrites.

Voltage-gated calcium channels (VGCCs)
* Are expressed almost exclusively and found at high density in synapses
* Their function is to get calcium into the terminal
* 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)

[Ca2+intracellular ] = ~nM[Ca2+extracellular ] = ~mM
1000? difference between the intracellular and extracellular [Ca2+]

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.

1. Action potential (axon)
Propagation of AP in the axon is primarily dependent on Na+ and K+ channels

2. Action potential (pre-synaptic terminal)
Depolarisation of terminal activates VGCCs, resulting in rapid entry of Ca2+

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

5. Vesicles are recycled

A change from -70 ? 0 mV

The channels are closed @ -70 mV

After the change to 0 mV there is a brief lag (0.5 ms) before the channels open

The black lines show single channel activity, they open/close at a high frequency
The probability of a channel being open (Popen) is never = 1
This means that the channel is closed for a long time whilst activated
Realistically the Popen = 0.3
Biological systems are not perfect

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.

This is because at this point the membrane is at -70 mV, which provides the strongest driving force within the time that the channels are open. This membrane potential is the furthest away from the equilibrium potential of Ca2+.

The two main neurotransmitters in the brain are…
Glutamate – predominantly excitatory (more likely for AP to occur)
GABA – predominantly inhibitory (less likely for AP to occur)
However there are some exceptions to this

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:

Neurotransmitters a co-transported into vesicles (not pumped)
The co-transporter relies on a high concentration of protons inside the vesicle (proton gradient)
A proton pump adds protons to the vesicle, using ATP as an energy source

Mechanisms are not well known, as neurons are difficult to study

* Synaptic vesicle is acidic (contains many H+)
* A sensor, GFP18 is quenched at low pH
* GFP18 is bound to a protein expressed in the vesicle
* GFP18 will be quenched when inside the vesicle
* Vesicle fuses causing protons to leave the vesicle, [H+] falls
* GFP18 is unquenched, it will fluoresce
* Fluorescence will continue until vesicle is restored and H+ are pumped inside it

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)

1. AP depolarised nerve terminal (< µs)
2. Opening of VGCCs (500 µs)
3. Calcium entry into nerve terminal (< µs)
4. Vesicle fusion (200 µs)
5. Neurotransmitter diffusion across cleft (1 µs) – very narrow cleft = fast diffusion

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.
The magnitude of the postsynaptic response (EPP) is proportional to [CaX]

Ca2+ Sensor – Possibilities:
The presynaptic terminal contains many proteins, many of which have 2+ Ca2+ binding domains
* Might explain the exponential relationship, requiring 2 Ca2+ to bind to function
Associated with vesicles and usually interact with the cytoskeleton is some way
e.g. Synapsins, synaptotagmin, synaptophysin

Synaptic Transmission – Presynaptic:

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).

7. Gating of ion channels
Binding of neurotransmitter to receptor can have multiple effects…
* A rapid opening of ion channels, resulting in a flux that changes the membrane potential of the postsynaptic neuron
* Release of second messengers in the postsynaptic neuron which modulates ion channels found in the membrane
* Activation of GTP-binding proteins that couple to ion channels in the membrane

The neuromuscular junction (NMJ):
A model system for understanding synaptic transmission
Very easy to experiment on

Acetyl choline (ACh) receptors are well studied. They respond to the release of ACh from motor neurons.
The end of a motor neuron is non-myelinated. The axon splits to form motor end plates that are in contact with the muscle cells and contain synapses.

1.Vesicles fuse with the synaptic membrane
* Up to 200 vesicles released / synapse
* Up to 5000 molecules ACh / vesicle
* The [ACh] rises to around 1mM in the synaptic cleft, taking < 100 ms

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

3.ACh binding causes Na+ to flow into the muscle cells
* AChR channels close and ACh rapidly dissociates (1-5 ms)

4.The influx of Na+causes a voltage change in the cell

5.Voltage gated calcium channels are activated within the muscle

6. Muscle contraction occurs

Synapses in the brain do work in a similar way to this however less is known about them
A dark mass can be seen on electron micrographs of the synaptic terminal. This is known as the active zone and is packed full of proteins that are involved with vesicular fusion. The vesicles can be seen clustered around this region. Actin docks the vesicles in an appropriate area.

*In the peripheral nervous system of mammals, ACh is used instead of glutamate as the excitatory transmitter
The ACh Receptor (AChR):
Studying ligand gated ion channels is difficult as they are membrane bound, making X-ray crystallography particularly difficult. When they are taken from a membrane their 3D structure is 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

* Heteromeric pentamer (?, ?, ?, ?, d)
* Each subunit is shown above – 4 TM domains (hydrophobic)
* M2 region lines the pore (5 in total)
* M2 is important as it determines how permeable the molecule is to an ion
* M2 is ?-helical
* Part of the cys-loop receptor family which also includes the GABA and glycine receptors
* Cys-loop contains disulphide bridges, an important structural element
* ACh ‘pocket’ is always between ? and ?
* ‘Cation (+ charged ion) permeable’ – less specific than the voltage gated channels
* Rings of ionic charge determine the ionic selectivity

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 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 (Popen is low)

In order to obtain a postsynaptic response, the transmitter needs to be removed quickly, to preventdesensitisation, which would occur from prolonged exposure to neurotransmitter.

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)

gs = amplitude of synaptic conductance
Nc = number of channels
? = single channel conductance
Popen = open probability

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

The duration of gs is determined by…
1. Transmitter profile
2. Channel kinetics (affinity of receptor)

An excitatory synaptic conductance (gs) results in a change in voltage (EPSP) that brings the neuron closer to the action potential threshold.

Ohm’s law can be used to predict the voltage change: V = I R
* How much current flows across the resistor (cell membrane)
* More membrane = more resistance

The duration of voltage change is determined by the membrane time constant
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
Molecular Cell Biology II Neuronal Signalling

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