Neuroscience - Classical neurotransmitter storage and release

Question 1

What is a quantal?

Answer 1

1 quantum of neurotrasmitter = content of 1 synaptic vesicle

Question 2

How many molecules approx are in 1 quantal?

Answer 2

5-10,000

Question 3

What does release of a single quantum cause?

Answer 3

A mini postsynaptic potential (can be spontaneous) - subthreshold for action potential

Question 4

What does the simultaneous release of many quanta cause?

Answer 4

A larger potential change sufficient to trigger an action potential

Question 5

What is synaptic delay?

Answer 5

Time between stimulation of the presynaptic neuron and the response in the postsynaptic cell

Question 6

What is the approximate synaptic delay time?

Answer 6

approx 0.4-5 milliseconds

Question 7

What is the quanta release in approx 1.5 milliseconds upon stimulation at the neuromuscular junction?

Answer 7

~300 quanta released in ~1.5 milliseconds

Question 8

How many ionotrophic receptors can each quanta activate?

Answer 8

2000 ionotrophic receptors

- permits 70 million ions to enter the post-synapse

Question 9

300 quanta release causes what kind of potential?

Answer 9

Excitatory postsynaptic potential (EPSP) of tens of mV eliciting an action potential

Question 10

How many quanta are released at central synapses and how many ion channels are activated?

Answer 10

~5-10 quanta

30 ion channels

Question 11

What is the ‘active zone’?

Answer 11

Site of neurotransmitter release

Question 12

What is the basic process of synaptic vesicle trafficking?

Answer 12

1) Vesicle release from endosome and neurotransmitter uptake
2) Trafficking to plasma membrane
3) Docking
4) Priming
5) Calcium signal causing exocytosis and neurotransmitter release
6) Endocytosis
7) Fusion to endosome

Question 13

What was the Nobel prize in Physiology or Medicine given for in 2013?

Answer 13

Discoveries of machinery regulating vesicle traffic

Question 14

What is the most abundant protein on the synaptic vesicle surface?

Answer 14

Synaptobrevin (VAMP)

Question 15

Name some other synaptic vesicle proteins

Answer 15

• Synaptotagmin

- Synapsin

Question 16

What experimental evidence show SNAREs roles in exocytosis?

Answer 16

1) Cleavage by bacterial toxin (botulinum toxin)
2) Loss of or mutations if SNARE proteins

Causes impaired locomotion and defective transmitter release

Question 17

What are the 3 SNARE proteins and where are they found?

Answer 17

1) Synaptobrevin (v-SNARE)
- found on the surface of synaptic vesicles

2) SNAP-25 (t-SNARE)
- Anchored to the cytosolic face of the pre-synaptic neuron

3) Syntaxin (t-SNARE)
- transmembrane protein of cytosolic face of pre-synaptic neuron

Question 18

What are the domain structures of the 3 SNARE proteins?

Answer 18

Synaptobrevin

• 1 SNARE motif
• Transmembrane domain

SNAP-25

• 2 SNARE motifs
• Anchored to membrane via palmitoylation of cysteines

Syntaxin

• 1 SNARE motif
• A Habc domain
• Flexible linker in between 2 above domains
• Transmembrane domain

Question 19

What kind of domains are SNARE motifs?

Answer 19

Coiled-coil (alpha helical) domains

Question 20

What are the 3 stages of SNARE complex assembly

Answer 20

1) Nucleation
- binding of v-SNARE and t-SNARE

2) Zippering
- Pulling the vesicle close to the membrane
- Induced by Ca2+

3) Fusion
- fusion of the vesicle membrane with the membrane of the pre-synaptic neuron

Question 21

How does membrane fusion occur?

Answer 21

• Energy from v and t SNARE coiling pulls membranes close together
• Water molecules are displaced from the hydrophilic faces of the membrane
• Mixing of lipids from from the facing leaflets of neighbouring membranes forms a connecting stalk (STALK FORMATION)
• The other leaflets of the membranes are pulled apart forming a new bilayer (HEMIFUSION)
• The force exerted by the fusion complex on the membrane anchors of the SNARE proteins disrupts the bilayer causing fusion
  (FUSION)

Question 22

What is Munc18 and what is its function?

Answer 22

• A SM protein family member
• Cytosolic protein that regulates fusion by binding to syntaxin in 2 ways
• Deletion/mutation prevents fusion

Question 23

In what 2 ways does Munc18 bind to syntaxin and what does this cause?

Answer 23

1) Binds to closed conformation of syntaxin
- Regulates fusion
- prevents release until appropriate signal

2) Binds to assembling SNARE complex via N-terminus of syntaxin
- essential for fusion

Question 24

What are the 3 active zone proteins and what are their general roles?

Answer 24

Munc13, RIM, and Rab3a

Roles in priming vesicles for release and in physically linking vesicles, Ca2+ channels and other presynaptic proteins in the active zone

Question 25

What is the structure of Munc13 and what are its roles?

Answer 25

- MUN domain interacts with SNAREs or SM proteins - C2A domain forms homodimer - Regulatory region composed of: > calmodulin-binding sequence > C1 domain > C2B domain that binds Ca2+ Essential for vesicle priming, regulated by Ca2+

Question 26

What is the structure and function of RIM?

Answer 26

- 2 alpha-helices that interact with vesicular Rab3a - Zinc finger that binds Munc13 C2a domain preventing homodimerisation - PDZ domain that binds Ca2+ channels - C2B domain Regulates Munc13 priming Tethers Ca2+ channels in proximity to docked vesicles Links various active zone proteins

Question 27

What is the structure and function of Rab3a?

Answer 27

- Small GTPase - Cycles between inactive GDP-bound cytosolic form and an active GTP-bound membrane associated form - Interacts with RIM and possible Munc18 - Assembles active zone compartments - Promotes vesicle docking by regulating vesicle-cytoskeleton interactions - May signal arrival of docked vesicle to RIM-Munc13

Question 28

How is SNARE complex dissembly carried out?

Answer 28

- NSF (an ATPase) binds to alpha-SNAP (A soluble NSF attachment protein - NSF uses energy from ATP hydolysis to unravel the coiled-coil interaction between v and t SNARE

Question 29

What occurs to synaptobrevin, SNAP-25 and syntaxin after SNARE dissembly

Answer 29

Synaptobrevin is recycled to the vesicle SNAP-25 and syntaxin remain in plamsa membrane

Question 30

What is the structure of synaptotagmin?

Answer 30

- N terminal integral membrane domain - large cytoplasmic domain with 2 C2 domains, C2A and C2B that bind Ca2+ - Interacts with memvrane phospholipids and SNAREs in a Ca2+ dependant manner

Question 31

What is the structure of complexin?

Answer 31

- small cytoplasmic protein | - central alpha-helical domain that binds to partially assembled SNARE complex

Question 32

Explain the model of synaptotagmin/complexin action

Answer 32

1) Complexin clamps partially assembled SNARE complex and superprimes the vesicle 2) Ca2+ bound synaptotagim interacts with SNARE complex and membrane phospholipids releasing complexin clamp 3) Synaptotagmin arrests the docked vesicles 4) Ca2+ binding to synaptotagmin pulls vesicles and membrane into tight apposition, leading to nucleation and zippering 5) Complexin binds and stabilises the SNARE complex

Question 33

What are the roles of the other vesicle protein, synapsin?

Answer 33

- Regulates numbers of vesicles in reserve and releasable pools - Implicated in synaptic plasticity and neurodevelopmental disorders