Lecture 10. Calcium Dependent Exocytosis Flashcards
What is on the extracellular side of the membrane ?
A local positive charge on the outside of the membrane
What is on the cytoplasmic side of the membrane ?
A local negative charge on the inside of the membrane
What does the charge separation at the membrane result in ?
Resting membrane potential
How does the potential arise ?
If the positive charges are allowed to, they will move back down across the gradient into the interior of the cell
What allows the potential to be held within the resting membrane potential ?
Holding the positive charges outside the cell against the concentration gradient
What are two things that contribute to the potential ?
- Chemical gradient
2. Electrical gradient
What are the proteins embedded in the membrane that influence the potential ?
- Potassium leak channels
2. Sodium potassium ATPase
What happens when potassium leak channels are accessible ?
Ions move out of the cell down their concentration gradient to deplete the extent of the potassium gradient between the inside and outside of the cell
What does sodium potassium ATPase do ?
It constantly hydrolyses ATP. It pumps out the three sodium ions and co-transports inwards two potassium ions. This transaction increases the positive charge outside the cell increasing the charge separation
What is the concentration of sodium outside the cell ?
120mM
What is the concentration of sodium inside the cell ?
16mM
What is the concentration of potassium outside the cell ?
63.8mM
What is the concentration of potassium outside the cell ?
3mM
What is the function of the pump ?
Maintains the concentration gradients and the charge separations and in that is stored the membrane potential
What allows the neuron to do work ?
The membrane potential
What causes the neuron to fire and release the neurotransmitter ?
Depolarisation
What is a huge amount of the neuron’s energy budget spent on ?
Pumping sodium out and potassium in
When do neurons typically depolarise ?
When gated ion channels open when a signal binds to these channels
What causes the channels to open and ions to flow in ?
The signal binds to the receptor on the gated ion channel which causes it to change its shape
What happens when a ligand binds to the channel ?
The ion channel is gated by the ligand and allows ion in causing sodium to rush in down the concentration and charge gradient
What does the resting membrane potential tend to be around ?
-65-70 mV
What happens when sodium ions rush into the negative cell ?
They depolarise the cell making it more positive. The resting membrane potential moves upwards towards zero because of the influx of positively charged sodium cations
What is a small depolarisation known as ?
A failed initiation that is insufficient to trigger the firing of an action potential
What are the two types of sodium gated channels ?
- Ligand gated sodium channels
2. Voltage gated sodium channels
What causes the first channels to open ?
The ligand binding and allows sodium to flow into the channel, changing the voltage of the membrane but not enough to cross the threshold
What causes the voltage gated ion channels to open ?
The change in voltage caused by the opening of ligand gated sodium channels, further depolarising the cell and allowing the potential to reach the threshold
What is end plate potential ?
When a neuron synapses onto a muscle cell
What is action potential propagation ?
Sodium moves into the cell and depolarises an area of the cell. It also opens further voltage gated channels nearby. This depolarisation process is repeated all the way down the axon in one direction only
What stops the depolarisation going backwards ?
Voltage gated potassium channels opening behind the sodium voltage gated ion channel
What is the concentration of potassium inside the cell verses outside the cell at the presynaptic terminal ?
400mM vs 20mM
What is the concentration of sodium outside the cell verses inside the cell ?
400mM vs 50mM
What happens when you block sodium channels with tetrodoxin ?
- Prevents sodium channels from opening
2. There is no presynaptic potential and no resulting post synaptic potential
What does TEA do ?
Shows the post synaptic potential does not repolarise
What happens if the two toxins are combined ?
There is less and less of the post synaptic potential to start with and doesn’t resolve at all
What happens if you block potassium channels ?
Neurotransmission is not prevented but the neurons fail to repolarise after the action potential has been triggered
What is the electrical trigger for depolarisation ?
Sodium
What is the chemical signal for neurotransmitter release ?
Calcium
What does sodium do ?
Changes the voltage and depolarisation opens the voltage gated calcium channels
What does calcium do ?
Triggers the changes in biochemistry of the vesicle allowing vesicles to fuse with them membrane and release contents (exocytosis)
What happens when sodium enters the channels in the region of the presynaptic terminal ?
It depolarises the entire terminal
Why do voltage gated calcium channels open ?
Because of the change in voltage caused by the depolarisation of the presynaptic terminal
What does calcium do ?
Enters the cell and facilitates neurotransmission
What is the synaptic vesicle cycle ?
- Vesicles have to be filled and cluster in the active zone.
- A small number have to dock at the active zone where they are primed for calcium dependent exocytosis.
- Calcium arrives and they must fuse with the membrane before recycling.
What do vesicles not do ?
Cross the synaptic cleft
What is an active zone ?
A specialised area that is thought to be the dock and release sites for synaptic vesicles
What are the proteins involved in the vesicular release of neurotransmitters ?
- SNARE proteins
- MUNC 18
- Complexin
- Synaptotagmin
What are the three SNARE proteins in the vesicular release of neurotransmitters ?
- Synaptobrevin- VAMP
- Syntaxin- 1
- SNAP-25
What does complexin do ?
Prevents certain synaptotagmin-SNARE binding interactions
What does synaptotagmin do ?
Cooperatively binds calcium and binds to both SNARE complex and phospholipid membrane to effect fusion pore opening
What is the SNARE complex made up of ?
Four alpha helix bundles formed between the vesicle and cell membrane
Where is synaptobrevin expressed ?
On the vesicle membrane
Where is syntaxin-1 and SNAP-25 expressed ?
Cell membrane
What is zippering ?
A process by which the alpha helices of the three SNARE proteins intertwine, resulting in the cell membrane effectively pulling the vesicle down to the membrane so the lipid bilayers are more tightly opposed
What happens at sites in the alpha helix in the SNARE complex ?
Bacterial endopeptidases can cleave all proteins
What does cleaving proteins in the SNARE complex achieve ?
Prevention of neurotransmission
What is the key intracellular sensor of calcium concentration ?
Synaptotagmin
Where is synaptotagmin superimposed ?
On the SNARE complex
What are the domains that synaptotagmin has ?
C2A and C2B
What is the function of C2A ?
Binds three calcium
What is the function of C2B ?
Binds two calcium
How does C2A and C2B bind ?
Cooperatively which increases the affinity for more calcium with each calcium bound
In the primed pre-fusion state, where does synaptotagmin bind ?
To the cell membrane and to the trimeric snare complex
What happens once calcium is bound to SNARE complex ?
Synaptotagmin can now bind the lipid membrane
What does binding of synaptotagmin to lipid membrane facilitate ?
Fusion and distortion of the membrane and the release of the neurotransmitter with the formation of the fusion pored
What are the steps involved in neurotransmitter release ?
- Sodium channels open
- Voltage changes - depolarisation
- Voltage dependent calcium channels open - calcium flows down concentration gradient
- Low affinity calcium sensors in active zone bind calcium
- Calcium dependent exocytosis
- Rapid turn off as calcium diffuses away from active zone
