Neuronal Signalling

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Molecular Cell Biology II Neuronal Signalling

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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

1.Action potentials rely on neurons being highly polarised

All cells do have an electrical potential, but it is less significant than in neurons2. Synaptic transmission enables the electrical signal to jump between cells

Intercellular communication3. 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 allcells 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, withATP 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.


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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. -70mVG

then there will be a driving force causing K+ to flow.

The amount of current that flows is equal to a parameter known as K

K is the conductance (a measure of permeability the ability of an ion to move across membrane)o permeability of channel=o conductance (K)

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.

o negative charge in / around the pore =o conductance

Current flow (I) =Kv (VM-Veq)

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

H

uring an action potential, all of these parameters will changeI

neuron is never at equilibrium, it is undergoing constant changes


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Molecular Cell BiologyII Neuronal Signalling

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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=-70mV

The - P 00mVequilibrium potential for K+ is never reached, due to the influence of other ions

Na+ equilibrium potential= +50mV Ca2+ can also have an effect

TheQ

ction Potential:

1. Resting membrane potential

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

2. R epolarisation

R epolarisation 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

TheQ Q

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 (-70mV)

b) Once the membrane has been depolarised to around-50mV, they start to open

Membrane is now very permeable to sodium ionsc) Membrane rapidly moves towards +50mV, trying to reach the equilibrium potential of sodium ions, but

doesnt reach this value

Ion channels dont stay open very long. They are inactivated soon after opening, despite being at theright voltage. It is important not to have the channel open for a long time.

At ~ +40mVthe K+ channels will open, dragging the voltage back.3. Repolarisation

Na+ channels get inactivated

Permeability to K+ increases

4. S yperpolarisation

The K+ channels remain open after repolarisation


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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 briefAHPs

o e.g. Interneurons in the Hippocampuso Need to encode very fast temporal information in K frequency range

Other cells do not require this, so have a longerAHPo e.g. dopaminergic neurons, involved in Parkisons diseaseo Broad, slow action potentials


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Myelination:

* 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)

T

unctions

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 theAP into the dendrites, the wrong way Myelin reduces the capacitance Not an issue in unmyelinated neurons as they are short


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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 (VU CCs)

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 o 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 VV CCs Voltage clamp technique allowed determination of time taken to open VW CCS after electrical signal

arrived (0.5 ms)

[Ca2+intracellular]= ~nM[Ca2+extracellular] = ~mM

1000v 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 ofAP in the axon is primarily dependent on Na + and K+ channels

2. Action potential (pre-synaptic terminal)

Depolarisation of terminal activates VW

CCs, 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


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[Page X ]

A change from Y X

p

m a

The channels are closed @ Y X

m a

After the change to

m a there is a brief lag (

.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 b 1

This means that the channel is closed for a long time whilst activated

Realistically the Popenb

.3

Biological systems are not perfect

There is also a slight lag (

.5 ms) when the membrane potential goes back to Y X

m a , 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 atY X

m a , 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+.


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The two main neurotransmitters in the brain are

Glutamate predominantly excitatory (more likely forAP to occur)

GABA predominantly inhibitory (less likely forAP 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 ~50nm 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 manyH+) A sensor, GFP18is quenched at low pH GFP18is bound to a protein expressed in the vesicle GFP18will be quenched when inside the vesicle Vesicle fuses causing protons to leave the vesicle, [H+] falls GFP18is unquenched, it will fluoresce Fluorescence will continue until vesicle is restored andH+ are pumped inside it

It is thought that full fusion does not occur, a kiss -and-run phenomena dominates instead

SynapticDelay:

There is a significant delay between the influx of calcium and the postsynaptic response (200c

s)

1. AP depolarised nerve terminal (

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Neuronal Signalling