Neuroscience - Classical neurotransmitter storage and release Flashcards
What is a quantal?
1 quantum of neurotrasmitter = content of 1 synaptic vesicle
How many molecules approx are in 1 quantal?
5-10,000
What does release of a single quantum cause?
A mini postsynaptic potential (can be spontaneous) - subthreshold for action potential
What does the simultaneous release of many quanta cause?
A larger potential change sufficient to trigger an action potential
What is synaptic delay?
Time between stimulation of the presynaptic neuron and the response in the postsynaptic cell
What is the approximate synaptic delay time?
approx 0.4-5 milliseconds
What is the quanta release in approx 1.5 milliseconds upon stimulation at the neuromuscular junction?
~300 quanta released in ~1.5 milliseconds
How many ionotrophic receptors can each quanta activate?
2000 ionotrophic receptors
- permits 70 million ions to enter the post-synapse
300 quanta release causes what kind of potential?
Excitatory postsynaptic potential (EPSP) of tens of mV eliciting an action potential
How many quanta are released at central synapses and how many ion channels are activated?
~5-10 quanta
30 ion channels
What is the ‘active zone’?
Site of neurotransmitter release
What is the basic process of synaptic vesicle trafficking?
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
What was the Nobel prize in Physiology or Medicine given for in 2013?
Discoveries of machinery regulating vesicle traffic
What is the most abundant protein on the synaptic vesicle surface?
Synaptobrevin (VAMP)
Name some other synaptic vesicle proteins
- Synaptotagmin
- Synapsin
What experimental evidence show SNAREs roles in exocytosis?
1) Cleavage by bacterial toxin (botulinum toxin)
2) Loss of or mutations if SNARE proteins
Causes impaired locomotion and defective transmitter release
What are the 3 SNARE proteins and where are they found?
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
What are the domain structures of the 3 SNARE proteins?
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
What kind of domains are SNARE motifs?
Coiled-coil (alpha helical) domains
What are the 3 stages of SNARE complex assembly
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
How does membrane fusion occur?
- 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)
What is Munc18 and what is its function?
- A SM protein family member
- Cytosolic protein that regulates fusion by binding to syntaxin in 2 ways
- Deletion/mutation prevents fusion
In what 2 ways does Munc18 bind to syntaxin and what does this cause?
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
What are the 3 active zone proteins and what are their general roles?
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
What is the structure of Munc13 and what are its roles?
- 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+
What is the structure and function of RIM?
- 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
What is the structure and function of Rab3a?
- 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
How is SNARE complex dissembly carried out?
- 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
What occurs to synaptobrevin, SNAP-25 and syntaxin after SNARE dissembly
Synaptobrevin is recycled to the vesicle
SNAP-25 and syntaxin remain in plamsa membrane
What is the structure of synaptotagmin?
- 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
What is the structure of complexin?
- small cytoplasmic protein
- central alpha-helical domain that binds to partially assembled SNARE complex
Explain the model of synaptotagmin/complexin action
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
What are the roles of the other vesicle protein, synapsin?
- Regulates numbers of vesicles in reserve and releasable pools
- Implicated in synaptic plasticity and neurodevelopmental disorders
