Modes of Synaptic Vesicle

release and recycling

 SNARE Hypothesis
• Synaptic vesicles have specific proteins that direct them to receptors on the
plasma membrane.
• The vesicle associated SNAP receptor (v-SNARE) in neurons is believed to be
synaptobrevin (VAMP).
• Target sites such as the plasma membrane (nerve terminal) would have
corresponding t-SNAREs (syntaxin and SNAP-25).

Coiled-coil
interactions.
Generic fusion mechanism involves
a tightening of the SNAREs to push
out H20.
 Synaptotagmin (P65) Ca2+ binding.

• It is a single polypeptide with one transmembrane spanning region

and a large cytoplasmic domain

• Protein kinase C like homology in carboxyl terminal, involved in

calcium and phospholipid binding. Injection of C2 peptide can
block release after the vesicle is docked.

• Forms tetramers with each binding calcium and cooperatively

consistent with 3-4 power of Ca dependence of release.

• In the absence of Ca2+ synaptotagmin may serve as a brake for

vesicle release. Ca2+ removes the brake.
Oligomerization of
synaptotagmin and its
binding to synaptic
protein partners occurs
over similar Ca2+
concentration
range as neurotransmitter
release.
Binding of Ca2+ and phospholipids by Synaptotagmins
( 1a, b, c) possibly promotes membrane fusion.
 Synapsins (Ia, Ib, IIa, IIb)
Vesicle associated proteins.
• Synapsin binds to cytoskeletal proteins including spectrin and
actin and enables closer docking of synaptic vesicles.
• The binding of synapsin to synaptic vesicle proteins is regulated
by phosphorylation,
• Binding of synapsin is weakened by phosphorylation - injecting
dephosphosynapsin blocks neurotransmitter release while CaMK
(active) increases it.
Synaptobrevin (VAMP-1 and 2, vesicle
associated membrane protein).
• Functions as a vesicle SNARE. Binds with t-
SNARE protein syntaxin and through a zippering
action brings vesicles close to membranes for
fusion

• Role in release is aided by use of toxins. Tetanus

toxin and botulinum B,D,F, and G (have protease
activity) cleave synaptobrevin selectively, and
blocks release.
 Soluble proteins.
• NSF - N-ethylmaleimide sensitive factor: isolated based on
its ability to restore function to a N-ethylmaleimide treated
golgi prep. NSF has ATP hydrolysis activity and is associated
with breaking down SNARE complexes after exocytosis.

• SNAPs-soluble NSF attachment proteins.

• Nsec-1/Munc-18 and munc 13, syntaxin binding proteins

likely bound to syntaxin when it is not in the SNARE
complex, involved in priming mechanism role unclear.
ATP is not actually required for release once
vesicles are docked, but is thought to break
down the SNARE complexes to promote
recycling.
 Plasma membrane proteins.
• Syntaxin putative t-SNARE, binds to N and P/Q-type
calcium channels (Sheng et al. 1994) and SNAP-25 and
synaptotagmin are critical for localizing Ca2+ channel near
release site.

• SNAP-25 peripheral membrane protein binds to syntaxin,

cleaved by botulinum toxin A and E. Forms a SNARE
complex with syntaxin and synaptobrevin.
Rizo and Sudhof 2002
Nature Rev. Neurosci.
 Synaptophysin
• Abundant synaptic vesicle protein of
unknown function.
Synthesis and Degradation
of
Neurotransmitters
 Acetylcholine Synthesis

Choline Acetyltransferase (ChAT)

Acetyl CoA + Choline Acetylcholine + CoA

+ CoA
 Degradation

Acetylcholine Esterase (AchE)

Acetylcholine Choline + Acetate

Choline Acetate
• AchE is present at the synaptic cleft

• Upon degradation of Ach, the Choline is taken up by

Na+/choline reuptake transporter

SARIN a nerve gas inhibits AchE and causes Ach accumulation

in the synaptic cleft, thereby constantly activating the Ach receptors.
This leads to muscle spasms and death due to neuromuscular
paralysis
Acetylcholine Receptors

• Nicotinic Receptor (Ionotropic)

• Muscarinic Receptor (Metabotropic)

Nicotinic Receptor
• Derives it name from its agonist Nicotine
• Ionotropic receptors, predominantly ligand gated
ion channels
• Heteropentameric in structure (2a, b, g, d
subunits)
• found at NM junctions of skeletal muscle,
neuronal junction of CNS and PNS
• Diffusion of Na+ and K+ across the receptor
causes depolarization, which then opens voltage-
gated Na+ channels.
• Binding of 2Ach molecules to 2a subunits causes
a conformational change to open the channel,
allowing Na+ and K+ to pass through and thereby
depolarizing the post-synaptic terminal.
• Curare is a potent antagonist for these receptors.
Muscarinic receptor
• Named after its agonist, Muscarine which is a poison from a
mushroom
• Not ion channels but G-protein coupled receptors, which activate
other ionic channels via a 2nd messanger eg IP3, DAG, cAMP.
• Predominant at the NM junctions of cardiac and smooth muscles,
and neuronal junction of brain and ANS.

 Classified as M1-M5 based on the 2nd messanger used

o M1, M4 & M5 upregulates IP3 and DAG

o M2 & M3 downregulate cAMP

Neuropharmacology of Acetylcholine
• a-Bungarotoxin- a peptide isolated from a snake venom, which binds
irreversibly to nicotinic AchR and causes paralysis

• Botulinum toxin- prevents Ach release by cleaving synaptobrevin and

syntaxin and thereby preventing vesicle fusion

• Black widow venom- stimulates Ach release by forming pores in the

pre-synaptic membrane to allow Ca2+ entry and thereby vesicle fusion.

• Atropine- blocks muscarinic receptors and is used to dilate pupil

• Curare-prevents nicotinic receptor transmission by irreversibly binding

to Ach receptors
 Myesthenia Gravis

• An auto-immune neuromuscular disorder because of production of

autoantibodies against acetylcholine receptors at the NM junction

• The autoantibodies block or destroy the receptors and

thereby muscle contraction is hindered. Hypothesized that certain
bacterial or viral infections promote the body’s immune system to
suddenly attack the AChE at NM junctions.

• Causes weakness and fatigue of the voluntary muscles that control eye
movement, facial expression, chewing, talking

• Generally treated with acetylcholine esterase inhibitors, neostigmine etc