Neurotransmitter Release Flashcards
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
Q
Functions and components of active zone cytomatrix
A
- Provide docking site for SVs and facilitate priming
- Recruit VGCCs
- Provide transsynaptic cell adhesion
Components:
- Synaptic cell adhesion proteins (neurexins)
- Scaffolding proteins (RIM, Munc13)
26
Q
Neurexins
A
- 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
Q
RIM
A
-modular scaffolding components with multiple functions:
- Tethering of SV via interaction with SV protein Rab3
- Support of SV priming via interaction with Munc13
- recruitment of VGCCs
28
Q
Munc13
A
- essential for NT release
- facilitate priming (SNARE complex formation)
- Munc13 activity regulated:
- calcium activates Munc13 and facilitates priming
- DAG also activates Munc13
29
Q
Sequence of events during clathrate mediated endocytosis
A
- Nucleation leads to assemble of a clathrin lattice on patches of PM
- Membrane invagination generates clathrin coated pits
- Fission of membrane to creat clathrin coated vesicle
- Uncoating: disassembly of clathrin coat
30
Q
Nucleation
A
- adaptor proteins (AP2 and AP180) bind to proteins to be endocytosis
- clathrin binds to adaptor proteins
- clathrin oligomerizes
31
Q
Invagination
A
- 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
Q
Membrane fission
A
- 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
Q
Mechanism of uncoating
A
- 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
Q
Actin in SV recycling
A
-2 roles:
- Transport of SV from endocytosis sites to active zone
- Retention of SV in resting pool to prevent them from reaching active zone
35
Q
Synapsins in SV recycling
A
- 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
Q
Stochastic nature of NT release
A
-synaptic transmission occur only with a certain NT release probability: Pr=# responses/# of trials
37
Q
Factors determining NT Pr
A
- 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
Q
Short term plasticity of NT release
A
-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
Q
Causes of short-term depression
A
- depletion of readily releasable pool of docked and primed SVs
- lower open time of Ca2+ channels, so less calcium influx
- receptor desensitization
40
Q
Short term facilitation
A
- following initial stimulation, basal calcium levels increase
- residual calcium can:
- increase SV docking
- accelerate SV priming
- facilitate SV fusion
