Vesicular neurotransmitter release

Question 1

what is NT release a highly specialised form of?

what are the 2 types of resolution?

what are the small packets called?

what is release triggered by?

what is the speed of the transduction of an electrical stimulus into vesicular release?

Answer 1

membrane fusion

spatial and temporal

quanta of NT

calcium-mediated fusion of vesicles

less than 1ms

Question 2

what did Katz discover about the amount of NT in a vesicle?

Answer 2

it was in defined quanta
- e.g. 10, 20, or 30 molecules
but not 12, or 27

Question 3

what do small synaptic vesicles contain?

what is a key feature of these transmitters?

describe the process of these SSVs

Answer 3

small molecule transmitters
e.g. dopamine, GABA

synthetic machinery resides in nerve terminal

1. vesicles synthesised in cell soma
2. trafficked up to nerve terminal
3. filled with small molecule transmitter

Question 4

what happens when SSVs reach the nerve terminal?

Answer 4

1. vesicles fuse with membrane
2. release quanta into synaptic cleft
3. vesicular machinery is re-used
4. vesicles are recycled and re-packaged with small molecules

Question 5

what type of response are small molecule transmitters involved in?

Answer 5

fast acting systems

- fast excitation and inhibition

Question 6

what do large dense-core vesicles (LDCVs) carry?

where is the synthetic machinery for these localised?

Answer 6

peptides
up to 90 amino acids

cell soma

Question 7

what is different between SSVs and LDCVs?

Answer 7

LDCVs are loaded at the cell soma

LDCVs are not recycled

LDCVs = 250nm
SSVs = 50nm

Question 8

what is the postsynaptic density?

Answer 8

highly complex structure just inside post-synaptic membrane

contains 100s of proteins involved in post-synaptic signalling and the coordination of post-synaptic events

Question 9

describe the process from action potential to ion channel opening on postsynaptic membrane

Answer 9

1. action potential propagation in presynaptic neurone
2. depolarisation opens Ca2+ channel
3. Ca2+ entry into synaptic knob
4. triggers release of NT by exocytosis
5. binding of NT to postsynaptic receptor
6. opening of specific ion channels

Question 10

why are there differences in NT release rates at different synapses?

give an example of a fast and slow synapse

Answer 10

response mechanism to a Ca2+ trigger

no. of vesicles in a release-ready state

rate and efficiency of replenishment

fast = inner ear synapse
= 1000s vesicles/ms

slow = fast cerebellar connection
= 3 vesicles/ms

Question 11

what are the 4 stages of NT release?

where are loaded SVs targeted too?

Answer 11

1. docking
2. priming
3. fusion
4. recycling

active zones

Question 12

describe docking

describe priming

Answer 12

membrane of vesicles tightly associates with plasma membrane
(others vesicles are in a reserve pool)

creation of a competent readily releasable pool of vesicles

Question 13

describe fusion

describe recycling

Answer 13

active fusion of vesicle membrane with plasma membrane to release vesicular contents when the local calcium reaches a threshold

recycling of vesicular membrane by endocytosis to form a new vesicle for transmitter filling

Question 14

what are the 2 forms of exocytosis?

Answer 14

full exocytosis
- fusion with membrane to release all contents

kiss and run

• vesicle partly fuses and releases of some of the contents
• then reseals

Question 15

what is synapsin?

Answer 15

a protein associated with the vesicle membrane

determines the release of vesicles from the reserve pool
-> anchors vesicles onto cytoskeleton at the reserve pool

Question 16

what is synaptobrevin (VAMPs)?

Answer 16

critical in fusion events

Question 17

what is synaptotagmin?

Answer 17

controls sensitivity of vesicles to local rise in ca2+

-> triggers fusion event

Question 18

what is Rab3 and what’s its role?

Answer 18

a small GTPase
- regulated by GTP-binding and hydrolysis

GTP-bound Rab3 (active) binds Rabphilin and the plasma membrane protein RIM

GDP-bound Rab3 (inactive) dissociates from vesicle

Question 19

what does fusion require?

what transport was this discovered to be essential for?

Answer 19

N-ethylmaleimide (NEM) sensitive factor (NSF)

together with SNAPs (soluble NSF attachment proteins)

ER-Golgi and intra-Golgi membrane transport

Question 20

what are SNAREs?

what do they include?

Answer 20

soluble-NSF-attachment proteins receptors

syntaxin
SNAP-25
synaptobrevin/VAMP

Question 21

describe the role of the fusion complex

Answer 21

VAMP attaches vesicle to fusion particle

SNAP25 + Syntaxin attach fusion particle to presynaptic membrane

Question 22

what is the functional role of NSF and SNAPs?

Answer 22

regulation of SNARe disassembly

Question 23

what do all membrane fusion secretory events involve?

Answer 23

formation of a helical coiled coil complex assembled from SNAREs

Question 24

which SNAREs are responsible for synaptic vesicle fusion?

Answer 24

Syntaxin 1
= presynaptic plasma membrane (integral)
- tSNARE

SNAP25
= presynaptic plasma membrane (Anchored)
- tSNARE

Synaptobrevin 2
= vesicular membrane (integral)
- vSNARE

Question 25

what do synaptic SNARES form?

Answer 25

a complex of 4 parallel helices

• Syntaxin and Synaptobrevin contribute 1 motif each
  and SNAP25 2 motifs

Question 26

how do clostridial toxins effect SNARE complexes?

Answer 26

tetanus and botulinum toxin:

act at Synaptobrevin

-> induces muscle paralysis

Question 27

how does Munc-18 act?

Answer 27

binds syntaxin 1
-> catalyses the step wise zippering of the SNAREs

designed to bind 4 helix bundles
-> pulls complex together

Question 28

what is the effect of having the 4 coiled coil complex?

Answer 28

gives rise to an inward force on the 2
membranes

-> pulls them together
= gives rise to a single fused membrane with a pore through the middle

Question 29

what happens when the vesicles first dock at the membrane?

Answer 29

enter a primed state
-> puts them into a hemifuse position

(undergo a partial fusion with nerve terminal membrane and held in that state until they switch to full fusion)

Question 30

how are vesicles held in a hemifused state?

Answer 30

Complexins clamps the SNARE complexes

• arrests fusion
  until synaptotagmi 1 can displace complexing from SNAREs in the presence of Ca2+

Question 31

describe the structure of Synaptotagmin 1

Answer 31

integral membrane protein on vesicles with 2 C-terminal C2 domains

calcium and phospholipid binding domains

Question 32

how does synaptotagmin work?

Answer 32

put a Ca2+-sensitive inhibitor constraint on exocytosis

when Ca2+ enters
-> synaptotagmin knocks complexin off