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Flashcards in Neurotransmitter Release Deck (40)
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1

NMJ to study NT release

-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

Miniature EPP

-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

Quantal nature of NT release at NMJ

-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

Evidence for NT storage in synaptic vesicles

-Katz observed accumulation of small vesicles at presynaptic terminals
-evidence: acetylcholine enriched in vesicles isolated from brain tissue by density gradient centrifugation (Whittaker)

5

NT quanta correspond to synaptic vesicles

-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

NT dependent on Calcium

-presynaptic injection of calcium chelators eliminate post synaptic potential

7

Inhibition of presynaptic VHCC by cone snail

-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

Characteristics of VGCC activation

-VGCC activate slowly in response to membrane depolarization
-delayed opening accounts for synaptic delay

9

Biogenesis of SV containing small-molecule NTs

-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

Loading of small molecule NT into SV

-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

Biogenesis of SV containing peptide NTs

-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

Evidence for local recycling of SV

-PM surface area needs to be held constant despite SV exocytosis: compensatory endocytosis

13

SV cycle

-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

SV pools

-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

Sequence of events leading to NT release

1. Docking: SV come in close proximity to PM
2. Priming: interaction between proteins in SV and PM
3. Fusion: of SV with PM is calcium dependent

16

SNARE complex

-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

Clostridial neurotoxins

-responsible for tetanus and botulism are highly specific proteases the block NT release by cleaving SNAREs

18

Munc18

-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

NSF and a-SNAP

-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

Synaptotagmin

-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

Synaptotagmin structure

-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

Synaptotagmin mechanism

-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

Synaptotagmin properties and implications for NT release

-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

Structure of active zone

-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