Neurotransmitter Release Flashcards
(40 cards)
1
Q
NMJ to study NT release
A
- NMJ used in 50s and 60s to study chemical transmission due to it being a large and accessible synapse
- motor neurons form large presynaptic terminal called end plates
- stimulation of Motorola nerve generates end plate potentials (EPP)
- EPP usually elicits AP in muscle
2
Q
Miniature EPP
A
- Katz studied changes in muscle membrane potential in the absence of motor nerve stimulation
- miniature EPP
- amplitude of mEPP is homogenous (around 0.5mv)
- mEPP are too big to represent potential change in response to singe acetylcholine receptor opening
3
Q
Quantal nature of NT release at NMJ
A
- motor nerve stimulation with low extracellular Ca2+: sometimes no EPP, sometimes very small
- amplitude of smallest EPP equals that of mEPP
- larger EPP are multiples of mEPP
- EPP made up of individual units elicited by exocytosis of a quantum of NTs
- mEPPs result from the spontaneous, AP independent release of one quantum of NT
4
Q
Evidence for NT storage in synaptic vesicles
A
- Katz observed accumulation of small vesicles at presynaptic terminals
- evidence: acetylcholine enriched in vesicles isolated from brain tissue by density gradient centrifugation (Whittaker)
5
Q
NT quanta correspond to synaptic vesicles
A
- freeze-fracture EM of NMJ (Heuser and Reese)
- NMJ stimulated then frozen and analyzed using electron microscopy
- vizualization of SV undergoing fusing
-4AP (potassium channel inhibitor) to increase NT release
- parallel recordings of EPPs to determine quantal content
- number of fusing synaptic vesicles and quantal content to correlate
- interpretation: exocytosis of individual synaptic vesicle leads to release of one quantum of NY
6
Q
NT dependent on Calcium
A
-presynaptic injection of calcium chelators eliminate post synaptic potential
7
Q
Inhibition of presynaptic VHCC by cone snail
A
- cone snail toxin causes paralysis
- omega-conotoxin black specific N-type voltage gated calcium channels
- calcium channels needed for NT release at NMJ
- funnel web spider toxin blocks P/Q VGCC which is necessary for NT release at central synapses
8
Q
Characteristics of VGCC activation
A
- VGCC activate slowly in response to membrane depolarization
- delayed opening accounts for synaptic delay
9
Q
Biogenesis of SV containing small-molecule NTs
A
- synthesis and uptake of small molecule NTs locally within presynaptic terminals
- either uptake of NT from extracellular space by plasma membrane transporters
- or uptake of precursors from extracellular space and local synthesis of precursors
10
Q
Loading of small molecule NT into SV
A
- NTs loaded into SV against electrochemical gradient by vesicular NT transporters
- secondary active transport: antiport of H+
- H+ gradient created by vesicular proton pump (uses ATP)
11
Q
Biogenesis of SV containing peptide NTs
A
- neuropeptides synthesize in the soma (ER->golgi)
- peptide filled large dense core vesicles are transported along microtubules via fast axonal transport
- neuropeptides do not undergo reuptake
- degraded by proteolytic enzymes
12
Q
Evidence for local recycling of SV
A
-PM surface area needs to be held constant despite SV exocytosis: compensatory endocytosis
13
Q
SV cycle
A
- endocytosis complete (10-20s) after exocytosis
- endocytosis vesicles bypass endosomes, becoming SVs
- recycled SV associate with PM and become fusion competent in approx 1 min
14
Q
SV pools
A
- readily releasable: SV immediately available for release (2-4%)
- reserve pool: SVs available for exocytosis but not immediate release (20%)
- resting pool: non-recycling SVs, 80%
15
Q
Sequence of events leading to NT release
A
- Docking: SV come in close proximity to PM
- Priming: interaction between proteins in SV and PM
- Fusion: of SV with PM is calcium dependent
16
Q
SNARE complex
A
- membrane fusion involves SNAREs
- SNARE complex bring negatively charged membrane into close apposition (energy required)
- SV membrane: synaptobrevin
- PM:syntaxin + SNAP25
- SNARE complex formation involves generation of energetically favourable alpha-helical bundle
17
Q
Clostridial neurotoxins
A
-responsible for tetanus and botulism are highly specific proteases the block NT release by cleaving SNAREs
18
Q
Munc18
A
- essential for evoked and spontaneous NT release
- binds syntaxin in closed conformation, unfolding it so it can interact with other SNARES
- binds to SNARE complex to facilitate SNARE complex mediated fusion directly
19
Q
NSF and a-SNAP
A
- disassemble SNARE complexes
- SNAREs are reused
- SNARE complexes very stable to chaperone is required to dissociate them
- NSF is the chaperone
- uses ATP as energy source
- NSF binds to complex via adapter protein a-SNAP
20
Q
Synaptotagmin
A
- calcium sensor for evoked release
- integral SV membrane protein with SNARE complex
- binds calcium
- AP eveoked fast release in mice lack functional synaptotagmin gene
- spontaneous release unaffected
21
Q
Synaptotagmin structure
A
- cytoplasmic c-terminus of synaptotagmin has 2 C@ domains that bind 4-5 calcium ions
- calcium binding cooperative: affinity for calcium initially low; rises with partial binding of Ca2+
- C2 domains bind phospholipids in a calcium dependent manner: affinity low in calcium free; high in calcium bound state
22
Q
Synaptotagmin mechanism
A
- binds to SNARE complex
- calcium binding leads to additional alectrostatic charges on C2 domains, causing them to bind negatively charged phospholipids in SV and PM
- membranes now in close apposition
- energetically favourable to fuse
23
Q
Synaptotagmin properties and implications for NT release
A
- low initial binding affinity of synaptotagmin for calcium; high calcium extrusion and buffering capacity of neurons means SV fusion only close to open calcium channels in calcium microdomains and nanodomains
- cooperative binding of calcium leads to super linear dependence of NT release on VGCC
24
Q
Structure of active zone
A
- docked SVs surrounded by filamentous material: active zone cytomatrix (large protein complex)
- active zone cytomatrix had modular structure at NMJ and possible CNS synapses
25
Functions and components of active zone cytomatrix
1. Provide docking site for SVs and facilitate priming
2. Recruit VGCCs
3. Provide transsynaptic cell adhesion
Components:
1. Synaptic cell adhesion proteins (neurexins)
2. Scaffolding proteins (RIM, Munc13)
26
Neurexins
- family of presynaptic cell adhesion proteins
- bind to families of postsynaptic cell adhesion proteins: neuroligins etc
- organize pre and postsynaptic specializations with scaffolding proteins such as CASK and PSD95
- implicated in neuro developmental disorders such as autism
27
RIM
-modular scaffolding components with multiple functions:
1. Tethering of SV via interaction with SV protein Rab3
2. Support of SV priming via interaction with Munc13
3. recruitment of VGCCs
28
Munc13
- essential for NT release
- facilitate priming (SNARE complex formation)
- Munc13 activity regulated:
- calcium activates Munc13 and facilitates priming
- DAG also activates Munc13
29
Sequence of events during clathrate mediated endocytosis
1. Nucleation leads to assemble of a clathrin lattice on patches of PM
2. Membrane invagination generates clathrin coated pits
3. Fission of membrane to creat clathrin coated vesicle
4. Uncoating: disassembly of clathrin coat
30
Nucleation
- adaptor proteins (AP2 and AP180) bind to proteins to be endocytosis
- clathrin binds to adaptor proteins
- clathrin oligomerizes
31
Invagination
- during oligomerization, clathrins triskelion structure facilitates invagination
- has spherical structure when many come together to help invagination
- integral membrane proteins such as Epson also promote membrane curvature
32
Membrane fission
- dynamin involved in membrane fission
- inhibition causes arrest of endocytosis at stage of invagination pits
- oligomerization of dynamin into stacks of rings around membrane stalk initiates budding
- Dynamin has GTPase activity: GTP hydrolysis causes tightening of rings, pinching off synaptic vesicles
33
Mechanism of uncoating
- Hsc70 is a chaperone needed to disassemble energetically favourable clathrin coat
- Hsc70 is an ATPase
- Hsc70 needs adaptor protein: Auxilin to bin to clathrin
34
Actin in SV recycling
-2 roles:
1. Transport of SV from endocytosis sites to active zone
2. Retention of SV in resting pool to prevent them from reaching active zone
35
Synapsins in SV recycling
- peripheral membrane proteins on SVs
- synapsins bind actin and may help anchor SV to cytoskeleton
- membrane association is phosphorylation dependent
- CaMKII or PKA causes dissociation from SVs so they can go to active zone
- proposed function: maintenance of reserve pool of SV
36
Stochastic nature of NT release
-synaptic transmission occur only with a certain NT release probability: Pr=# responses/# of trials
37
Factors determining NT Pr
- size of readily releasable SV pool (RRP)
- RRP determined by # of SV that can dock at active zone, and fraction of docked SVs that are primed
- individual SVs in RRP display different likelihood of AP triggered fusion
- differences in presynaptic calcium currents
- differences in spatial coupling of calcium influx and primed SVs
38
Short term plasticity of NT release
-in response to repetitive stimulation, NT release can facilitate or depress
- high Pr synapses often depress
- low Pr synapses often facilitate
- many show both
39
Causes of short-term depression
- depletion of readily releasable pool of docked and primed SVs
- lower open time of Ca2+ channels, so less calcium influx
- receptor desensitization
40
Short term facilitation
- following initial stimulation, basal calcium levels increase
- residual calcium can:
- increase SV docking
- accelerate SV priming
- facilitate SV fusion
