Week 5 (neurotransmitter release) physiology Flashcards
neurotransmitter gradient in neuron vs out
glutamate: 1μM outside, 10mM inside (10000x)
active process that maintains this gradient.
formation of neurotransmitter gradient (in/out cell)
Through active process:
Utilising Na+ gradient: eg. glutamate and noradrenaline uses Na+ coupled transporters to be transported inwards.
formation of neurotransmitter gradient (in cell/vesicle)
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
protonmotive force
A pH gradient (Highly acidic)
and
A electrical gradient (membrane potential) (Highly +vely charged inside)
blocking vacuolar ATPase
Drug: Bafilomycin A1
no uptake of any neurotransmitter into vesicle
no protonmotive force
inserting NH3 into vesicle
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.
2 pools of neurotransmitter vesicles inside neuron
- 90% are the reserve pool
- 10% are the readily releasable pool (waiting for Ca2+ influx)
reserve pool synaptic vesicles are held by…
connected to actin cytoskeleton via synapsin
synapsin KO consequence (mice model)
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.
What regulates synapsin activity - allowing vesicle translocation
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
-
PKA (phosphorylation): loses affinity to phospholipid
synapsin detaches from vesicle
terminal stimulation (Ca2+ influx) and protein kinase activity
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.
3 SNAREs (SNAp REceptors)
integral to vesicle membrane: synaptobrevin
integral to plasma membrane: syntaxin
loosely bound to integral plasma membrane: SNAP-25
V-SNARE (vesicle)
synaptobrevin
t-SNARE (target)
syntaxin and SNAP-25
docking process
vSNARE binds to tSNARE to form 7S SNARE complex
NSF (ATPase)
after fusion - breaking SNARE complex:
SNARE complex (cis is more breakable, trans form is very stable)
using ATP hydrolysis energy:
complex disassembly
alpha SNAP
brings NSF to attach to SNARE complex
SNARE complex + alpha SNAP and NSF
20S SNARE complex
must/essential proteins for synaptic vesicle fusion
v/tSNAREs:
tetanus toxin and all Botulinum toxins
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
SNARE complex is thermodynamically favourable
The complex is resistant to:
- proteases - toxins
- boiling
- detergents that interrupt protein-protein interaction
Are SNAREs the minimum machinery required for membrane fusion?
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.
active zone - specificity (Ca2+ channels)
Ca2+ channels bind to syntaxin (tSNARE)
via a synprint site.
synatic vesicle exocytosis (reason for high conc Ca2+ channels)
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
Synaptotagmin - Requirements of nerve terminal protein to be Ca2+ trigger
- 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
- 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+
- Cooperative binding with Ca2+ — 4 binding sites with different affinities
- Bind to other proteins in a Ca2+-dependent manner — trigger action
synaptotagmin (Ca2+ binding domains)
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.
synaptotagmin KO (mice model)
consequence: inhibition of Ca2+ stimulated neurotransmitter release
synaptotagmin KO complication and reversal effect
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)
Caged Ca2+ and UV
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
vesicle priming
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.
fusion clamp vs delayed zippering hypothesis
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.
