Lecture 10. Calcium Dependent Exocytosis

Question 1

What is on the extracellular side of the membrane ?

Answer 1

A local positive charge on the outside of the membrane

Question 2

What is on the cytoplasmic side of the membrane ?

Answer 2

A local negative charge on the inside of the membrane

Question 3

What does the charge separation at the membrane result in ?

Answer 3

Resting membrane potential

Question 4

How does the potential arise ?

Answer 4

If the positive charges are allowed to, they will move back down across the gradient into the interior of the cell

Question 5

What allows the potential to be held within the resting membrane potential ?

Answer 5

Holding the positive charges outside the cell against the concentration gradient

Question 6

What are two things that contribute to the potential ?

Answer 6

1. Chemical gradient

2. Electrical gradient

Question 7

What are the proteins embedded in the membrane that influence the potential ?

Answer 7

1. Potassium leak channels

2. Sodium potassium ATPase

Question 8

What happens when potassium leak channels are accessible ?

Answer 8

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

Question 9

What does sodium potassium ATPase do ?

Answer 9

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

Question 10

What is the concentration of sodium outside the cell ?

Answer 10

120mM

Question 11

What is the concentration of sodium inside the cell ?

Answer 11

16mM

Question 12

What is the concentration of potassium outside the cell ?

Answer 12

63.8mM

Question 13

What is the concentration of potassium outside the cell ?

Answer 13

3mM

Question 14

What is the function of the pump ?

Answer 14

Maintains the concentration gradients and the charge separations and in that is stored the membrane potential

Question 15

What allows the neuron to do work ?

Answer 15

The membrane potential

Question 16

What causes the neuron to fire and release the neurotransmitter ?

Answer 16

Depolarisation

Question 17

What is a huge amount of the neuron’s energy budget spent on ?

Answer 17

Pumping sodium out and potassium in

Question 18

When do neurons typically depolarise ?

Answer 18

When gated ion channels open when a signal binds to these channels

Question 19

What causes the channels to open and ions to flow in ?

Answer 19

The signal binds to the receptor on the gated ion channel which causes it to change its shape

Question 20

What happens when a ligand binds to the channel ?

Answer 20

The ion channel is gated by the ligand and allows ion in causing sodium to rush in down the concentration and charge gradient

Question 21

What does the resting membrane potential tend to be around ?

Answer 21

-65-70 mV

Question 22

What happens when sodium ions rush into the negative cell ?

Answer 22

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

Question 23

What is a small depolarisation known as ?

Answer 23

A failed initiation that is insufficient to trigger the firing of an action potential

Question 24

What are the two types of sodium gated channels ?

Answer 24

1. Ligand gated sodium channels

2. Voltage gated sodium channels

Question 25

What causes the first channels to open ?

Answer 25

The ligand binding and allows sodium to flow into the channel, changing the voltage of the membrane but not enough to cross the threshold

Question 26

What causes the voltage gated ion channels to open ?

Answer 26

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

Question 27

What is end plate potential ?

Answer 27

When a neuron synapses onto a muscle cell

Question 28

What is action potential propagation ?

Answer 28

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

Question 29

What stops the depolarisation going backwards ?

Answer 29

Voltage gated potassium channels opening behind the sodium voltage gated ion channel

Question 30

What is the concentration of potassium inside the cell verses outside the cell at the presynaptic terminal ?

Answer 30

400mM vs 20mM

Question 31

What is the concentration of sodium outside the cell verses inside the cell ?

Answer 31

400mM vs 50mM

Question 32

What happens when you block sodium channels with tetrodoxin ?

Answer 32

1. Prevents sodium channels from opening | 2. There is no presynaptic potential and no resulting post synaptic potential

Question 33

What does TEA do ?

Answer 33

Shows the post synaptic potential does not repolarise

Question 34

What happens if the two toxins are combined ?

Answer 34

There is less and less of the post synaptic potential to start with and doesn't resolve at all

Question 35

What happens if you block potassium channels ?

Answer 35

Neurotransmission is not prevented but the neurons fail to repolarise after the action potential has been triggered

Question 36

What is the electrical trigger for depolarisation ?

Answer 36

Sodium

Question 37

What is the chemical signal for neurotransmitter release ?

Answer 37

Calcium

Question 38

What does sodium do ?

Answer 38

Changes the voltage and depolarisation opens the voltage gated calcium channels

Question 39

What does calcium do ?

Answer 39

Triggers the changes in biochemistry of the vesicle allowing vesicles to fuse with them membrane and release contents (exocytosis)

Question 40

What happens when sodium enters the channels in the region of the presynaptic terminal ?

Answer 40

It depolarises the entire terminal

Question 41

Why do voltage gated calcium channels open ?

Answer 41

Because of the change in voltage caused by the depolarisation of the presynaptic terminal

Question 42

What does calcium do ?

Answer 42

Enters the cell and facilitates neurotransmission

Question 43

What is the synaptic vesicle cycle ?

Answer 43

1. Vesicles have to be filled and cluster in the active zone. 2. A small number have to dock at the active zone where they are primed for calcium dependent exocytosis. 3. Calcium arrives and they must fuse with the membrane before recycling.

Question 44

What do vesicles not do ?

Answer 44

Cross the synaptic cleft

Question 45

What is an active zone ?

Answer 45

A specialised area that is thought to be the dock and release sites for synaptic vesicles

Question 46

What are the proteins involved in the vesicular release of neurotransmitters ?

Answer 46

1. SNARE proteins 2. MUNC 18 3. Complexin 4. Synaptotagmin

Question 47

What are the three SNARE proteins in the vesicular release of neurotransmitters ?

Answer 47

1. Synaptobrevin- VAMP 2. Syntaxin- 1 3. SNAP-25

Question 48

What does complexin do ?

Answer 48

Prevents certain synaptotagmin-SNARE binding interactions

Question 49

What does synaptotagmin do ?

Answer 49

Cooperatively binds calcium and binds to both SNARE complex and phospholipid membrane to effect fusion pore opening

Question 50

What is the SNARE complex made up of ?

Answer 50

Four alpha helix bundles formed between the vesicle and cell membrane

Question 51

Where is synaptobrevin expressed ?

Answer 51

On the vesicle membrane

Question 52

Where is syntaxin-1 and SNAP-25 expressed ?

Answer 52

Cell membrane

Question 53

What is zippering ?

Answer 53

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

Question 54

What happens at sites in the alpha helix in the SNARE complex ?

Answer 54

Bacterial endopeptidases can cleave all proteins

Question 55

What does cleaving proteins in the SNARE complex achieve ?

Answer 55

Prevention of neurotransmission

Question 56

What is the key intracellular sensor of calcium concentration ?

Answer 56

Synaptotagmin

Question 57

Where is synaptotagmin superimposed ?

Answer 57

On the SNARE complex

Question 58

What are the domains that synaptotagmin has ?

Answer 58

C2A and C2B

Question 59

What is the function of C2A ?

Answer 59

Binds three calcium

Question 60

What is the function of C2B ?

Answer 60

Binds two calcium

Question 61

How does C2A and C2B bind ?

Answer 61

Cooperatively which increases the affinity for more calcium with each calcium bound

Question 62

In the primed pre-fusion state, where does synaptotagmin bind ?

Answer 62

To the cell membrane and to the trimeric snare complex

Question 63

What happens once calcium is bound to SNARE complex ?

Answer 63

Synaptotagmin can now bind the lipid membrane

Question 64

What does binding of synaptotagmin to lipid membrane facilitate ?

Answer 64

Fusion and distortion of the membrane and the release of the neurotransmitter with the formation of the fusion pored

Question 65

What are the steps involved in neurotransmitter release ?

Answer 65

1. Sodium channels open 2. Voltage changes - depolarisation 3. Voltage dependent calcium channels open - calcium flows down concentration gradient 4. Low affinity calcium sensors in active zone bind calcium 5. Calcium dependent exocytosis 6. Rapid turn off as calcium diffuses away from active zone