Week 5 (neurotransmitter release) physiology

Question 1

neurotransmitter gradient in neuron vs out

Answer 1

glutamate: 1μM outside, 10mM inside (10000x)
active process that maintains this gradient.

Question 2

formation of neurotransmitter gradient (in/out cell)

Answer 2

Through active process:
Utilising Na+ gradient: eg. glutamate and noradrenaline uses Na+ coupled transporters to be transported inwards.

Question 3

formation of neurotransmitter gradient (in cell/vesicle)

Answer 3

In the cytosol, the vacuolar ATPase will pump H+ into the vesicle creating a protonmotive force.

Glutamate (-ve): attracted to highly positive membrane potential, uses membrane potential gradient.
Noradrenaline: antiport, uses pH gradient

Question 4

protonmotive force

Answer 4

A pH gradient (Highly acidic)
and
A electrical gradient (membrane potential) (Highly +vely charged inside)

Question 5

blocking vacuolar ATPase

Answer 5

Drug: Bafilomycin A1
no uptake of any neurotransmitter into vesicle
no protonmotive force

Question 6

inserting NH3 into vesicle

Answer 6

What’s affected?
Noradrenaline cannot be uptake by vesicles because pH gradient is eliminated by NH3 binding to H+.

NH4+, this does not affect Glu- uptake.

Question 7

2 pools of neurotransmitter vesicles inside neuron

Answer 7

1. 90% are the reserve pool
2. 10% are the readily releasable pool (waiting for Ca2+ influx)

Question 8

reserve pool synaptic vesicles are held by…

Answer 8

connected to actin cytoskeleton via synapsin

Question 9

synapsin KO consequence (mice model)

Answer 9

no reserve pool of synpatic vesicles could be seen.
nothing to hold the vesicles onto the actin cytoskeleton

Since no reserve:
Greatly reduced neurotransmitter release after repetitive stimuli.

Question 10

What regulates synapsin activity - allowing vesicle translocation

Answer 10

Synapsin is a phosphoprotein.
1. not phosphorylated: binds to actin and phospholipid
2. CaMKII (calmodulin protein kinase - phosphorylation): loses affinity to actin
synapsin loses connection to actin cytoskeleton

3. PKA (phosphorylation): loses affinity to phospholipid
   synapsin detaches from vesicle

Question 11

terminal stimulation (Ca2+ influx) and protein kinase activity

Answer 11

PKA and CaMKII are both Ca2+ sensitive:
1. PKA: activated through Ca2+ - cAMP pathway
2. CaMKII undergoes conformational change after Ca2+ binding to become active.

Question 12

3 SNAREs (SNAp REceptors)

Answer 12

integral to vesicle membrane: synaptobrevin
integral to plasma membrane: syntaxin
loosely bound to integral plasma membrane: SNAP-25

Question 13

V-SNARE (vesicle)

Answer 13

synaptobrevin

Question 14

t-SNARE (target)

Answer 14

syntaxin and SNAP-25

Question 15

docking process

Answer 15

vSNARE binds to tSNARE to form 7S SNARE complex

Question 16

NSF (ATPase)

Answer 16

after fusion - breaking SNARE complex:
SNARE complex (cis is more breakable, trans form is very stable)

using ATP hydrolysis energy:
complex disassembly

Question 17

alpha SNAP

Answer 17

brings NSF to attach to SNARE complex

Question 18

SNARE complex + alpha SNAP and NSF

Answer 18

20S SNARE complex

Question 19

must/essential proteins for synaptic vesicle fusion

Answer 19

v/tSNAREs:

Question 20

tetanus toxin and all Botulinum toxins

Answer 20

toxins = selective proteases
cleave the SNAREs and inhibit synaptic vesicle exocytosis

toxins cleave one of the SNAREs: eg. syntaxin is cleaved by BoNT C

• Heavy chain: facilitates receptor-mediated endocytosis on plasma membrane
• light chain: metalloproteases that perform the cleavage action

Question 21

SNARE complex is thermodynamically favourable

Answer 21

The complex is resistant to:
- proteases - toxins
- boiling
- detergents that interrupt protein-protein interaction

Question 22

Are SNAREs the minimum machinery required for membrane fusion?

Answer 22

artificial membrane and SNAREs insertion:
fluorescence is shown if membrane fuses
only the 3 SNAREs are added on artificial membranes.

Over time: with only 3 SNAREs, fluorescence is shown.
EVENTHOUGH very slow, it still works.

Question 23

active zone - specificity (Ca2+ channels)

Answer 23

Ca2+ channels bind to syntaxin (tSNARE)
via a synprint site.

Question 24

synatic vesicle exocytosis (reason for high conc Ca2+ channels)

Answer 24

fast and ATP-independent but:
REQUIRES a huge amount of Ca2+ for stimulation.
Only this microdomain will have this high concentration temporarily
The high concentration of Ca2+ channels is at the docking site, ensuring fast exocytosis

Question 25

Synaptotagmin - Requirements of nerve terminal protein to be Ca2+ trigger

Answer 25

1. Close proximity with active zone - Ca2+ influx - (outcompetes EGTA - relatively slow binding speed - by being in proximity to the active zone) —integral synaptic vesicle protein
2. Bind Ca2+ with low affinity (artificial BAPTA outcompetes synaptotagmin in Ca2+ binding due to high affinity) — binds to Ca2+ with a Kd of 20 microM Ca2+
3. Cooperative binding with Ca2+ — 4 binding sites with different affinities
4. Bind to other proteins in a Ca2+-dependent manner — trigger action

Question 26

synaptotagmin (Ca2+ binding domains)

Answer 26

C2A and C2B: 2 calcium binding domains with 4 binding sites (binding of 1 will increase affinity of another)
C2A (Ca2+ bound) : will also bind to phospholipids
C2B (Ca2+ bound) : will bind to phospholipids and tSNAREs

(Ca2+ bound) C2 domains bind to plasma membrane and bend it toward the vesicle.

Question 27

synaptotagmin KO (mice model)

Answer 27

consequence: inhibition of Ca2+ stimulated neurotransmitter release

Question 28

synaptotagmin KO complication and reversal effect

Answer 28

will s. KO affect other genes that may be regulating vesicle exocytosis?
- Test by putting wild type s. back – full restoration
- Test by putting s. with lower Ca2+ affinity back – level of Ca2+ required to stimulate vesicle exocytosis is increased due to lower affinity. (shows that neurotransmitter release is dependent on the Ca2+ binding ability of synaptotagmin)

Question 29

Caged Ca2+ and UV

Answer 29

Allow uniform increase of calcium concentration in the neuron.
Above 20 microM of calcium:
exponential increase of exocytosis (>1 Ca2+ binding site) cooperative nature.

Above 278microM of calcium:
maximal exocytosis - saturated binding

Question 30

vesicle priming

Answer 30

Priming allows higher synchrony and less of a gap between depolarisation and neurotransmitter release.

Complexin binds to the SNARE complex and clamps it in an activated state of zippering (but blocks it from fully zippering).

During Ca2+ binding (influx), synaptotagmin will displace complexin and allow vesicle fusion to happen - releasing the neurotransmitters.

Question 31

fusion clamp vs delayed zippering hypothesis

Answer 31

fusion clamp: complexin binds to the partially assembled SNARE complex, stabilizing it in a ‘clamped’ state. This state prevents the complete zippering of the SNARE complex

delayed zippering: complexin doesn’t completely inhibit the zippering of the SNARE complex but rather delays it, keeping it in a partially zippered state, calcium influx, complexin’s inhibitory effect is overcome.