SYNAPTIC TRANSMISSION: UNIT 2

I.

Lecture 7
a. Calcium Regulation and Transmitter Release
i. Scheme of AP invading terminal and steps leading to secretion:
1. Action potential/nerve terminal depolarization
2. Activation of voltage-gated Ca++ channels
3. Calcium enters down a strong concentration gradient
4. Calcium triggers transmitter release
5. Exocytosis of transmitter
6. Transmitter crosses the synaptic cleft
7. Postsynaptic conductance change
8. Postsynaptic action potential
ii. Synaptic delay: the time between the presynaptic action potential and
postsynaptic effect, taking about 1 msec. Half of this time is the first few
steps until voltage-gated Calcium channels open
b. Regulation by Calcium
i. One of the first regulatory mechanisms to be studied for transmitter
release was identified by Katz in the early 60s
ii. Normal Ca++ is very low inside cells, and relatively high outside of cells
1. Normal IC = 10-100 nM
2. Normal EC = 2 mM (2x10-8 vs. 2x10-3 five orders of magnitude)
iii. Major difference is how low the IC Ca++ is this allows for calcium to be
a signaling ion
iv. During AP activity, Katz did several classical experiments that led him to
hypothesize that Ca++ entered the nerve terminal and regulated vesicular
release:
1. NMJ in bath remove Ca++ from bath no transmitter release,
although AP and postsynaptic sensitivity remained the same
2. Add ionic blocker (Mg++ or Cd++) also prevents release
blocks Ca++ flux by competition
3. Conclusion: calcium is necessary for transmitter release
v. Since then, new experiments have shown that Ca++ is also sufficient to
trigger release:
1. Calcium ionopores (not voltage gated) causes release
2. Loading terminals with caged calcium liberation induces
transmitter release
3. Calcium liposomes also triggers release
vi. Calcium is both necessary and sufficient to causes transmitter release,
but it is NOT dependent on calcium entry from ion channels; just need to
increase intracellular calcium
c. Calcium Homeostasis
i. The concentration difference across the neuron membrane is very large.
Cells use Ca++ for many IC functions normally cells drop it well below
the activation threshold for these many events
ii. How cells maintain low IC [Ca]

1. Transport Systems
a. Ca-Mg ATPase
i. Activated by IC calcium
ii. High affinity for calcium (<300nM)
iii. Mg is need as a cofactor for ATP binding
iv. 1 calcium excreted for every ATP hydrolyzed
b. Na-Ca Exchanger
i. Activated by IC calcium
ii. Lower affinity for calcium
iii. Driven by sodiums electrochemical gradient
iv. 1 Ca++ out for every 3 Na++ in
1. Electrogenic: cellular depolarization of +1
v. Not as many of these in neurons as sodiumpotassium pumps; thus, does not have a rapid or
potent effect on the membrane potential
2. Buffers
a. Although there is very low free ionic calcium in neurons,
they do contain a lot of calcium that is bound up by buffers
i. ER (regulated), mitochondria (rapidly absorbs, then
slowly dribbles out), proteins
iii. Affecting Calcium Buffering
1. Add calcium to nerve terminal, see effects on transmitter release
with:
a. EGTA: high affinity, low speed of binding no effect
b. BAPTA: high affinity, fast speed of binding blocks
release
2. Shows that a fast buffer is needed to grab calcium after it enters the
nerve terminal and before it binds to the secretory apparatus. Thus,
calcium entry and release apparatus must be very close to one
another
iv. Squid Giant Synapse
1. Fura measurement of calcium concentration shows a gradient from
closely opposed membrane (pre and post) into nerve terminal
following high frequency stimulation (synthesized calcium buffers
change fluorescence when they bind calcium)
d. Calcium Concentration Needed for Transmitter Release
i. Using caged calcium and various buffered calcium concentrations,
investigators have shown that they can only mimic the nerve stimulated
release with 10-100 uM inside the nerve terminal
ii. So the Fura dye visualization in the squid synapse was just showing us the
flood of calcium spreading through the terminal; we would get a better
image of the relevant calcium with a different dye. To really see the
calcium that the release apparatus uses, we need to use a low affinity dye
that only lights up when calcium reaches the uM range.
1. Now see resting flickers that increase in frequency when you
stimulate these have been called microdomains

II.

a. Microdomains hypothesized to be the Ca++ just under the

calcium channel that triggers transmitter release
2. Synaptic delay and effects of exogenous buffers predict that
calcium channels should occur very close to release apparatus
iii. Robataille and Cohen imagined presynaptic calcium channels and
postsynaptic ACh receptors in perfect alignment
iv. Often synapses signal with burst of action potentials. Bursts are high
enough frequency that there is some residual calcium in the nerve terminal
between each AP that builds up
e. Short-Term Plasticity
i. Most a balance between residual calcium effects that increase release, and
vesicle depletion effects that decrease release
1. Paired pulse facilitation: when two stimuli are delivered in rapid
succession, the second stimuli evokes a larger response than the
first
2. Tetanic potentiation: during a train of multiple stimuli at high
frequency, there is a gradual increase in the amount of
neurotransmitter released
3. Post-tetanic potentiation: after a train of stimuli has ended, there
is an increase in the amount of transmitter release with single
stimuli at low frequency for some time
ii. Not necessarily due to residual free ionic Ca++; good buffers to handle
this free calcium, but bound calcium is often in equilibrium with these
buffers, or is moved out of sponges later (e.g. mitochondria dribble it
out gradually) to be pumped out by Ca-Mg ATPase
Calcium Channels
a. Measurements of Calcium Current in Nerve Terminals
i. Need model system that allows one to patch a nerve terminal
1. Squid giant synapse
2. Chick calyx
3. Auditory calyx of Held
4. Xenopus nerve muscle culture
b. Calcium Current-Voltage Relationship
i. Similar to Na+ due to similar DF and activation curves
ii. Tail current: deactivation of the micro and macro current scale. Not seen
much with sodium channels because they inactivate so fast, but voltagegated calcium channels are sluggish, so they dont close before the driving
force increases.
iii. Calcium entry during an action potential; balance of influences leads to a
tail current during an action potential waveform
1. Recorded at -40 mV after depolarization
iv. Given that Ca++ is the trigger for exocytosis, what is the relationship
between Ca++ entry into the terminal and NT release?
1. Initially, just change Ca++ outside and see how transmitter release
changes

a. Release is very sensitive to Calcium; as soon as you a drop

a little, transmitter releases drops also
2. Equation to describe relationship:
a. Fold change in postsynaptic measure of release = Fold
change in [Ca++] raised to the 3-4
b. A 3rd or 4th order relationship
c. On a log-log plot, you get a straight line with a slope of 3-4
i. Ex. 0.15 to 0.3 mM change in calcium
concentration (2 fold) alters postsynaptic release
from 0.25 mV to 4 mV (16 fold) (24 = 16)
3. What does this mean?
a. Important for modulation of synapses; a small change in
calcium current can have a big effect on transmitter
release; easier for plasticity to occur
4. How does one come about this?
a. Calcium must bind to multiple vesicle-associated binding
sites in a cooperative manner for release to occur
i. Either one protein with multiple sites, or several
different proteins that bind calcium, or something in
between
b. It be that one calcium ion binds to each vesicle to trigger
NT release, or else there would be a linear relationship
c. Calcium Channel Structure, Types, and Properties
i. Voltage-gated calcium channels have been localized to transmitter release
sites and open following action potential depolarization
ii. Basic structure
1. 4 domains, S4 voltage sensor, and p-loop
2. Intracellular binding sites on cytoplasmic amino acids for calcium
3. Alpha 1 subunit pore forming
4. Beta subunit auxiliary subunit; modulates channel function and
facilitates movement from Golgi to membrane
5. Beta-Gamma subunit modulatory protein; alters how much
calcium comes in
iii. Heavily regulated
1. Fine tuning for transmitter release because so many binding sites
within the channel
iv. More than 1 type of VG Ca++ channel
1. There are at least 5 pharmologic types of and about the same
number of gene families that code for pore forming alpha 1 subunit
2. There are auxiliary subunits (alpha 2, gamma, beta) that can
combine with pore-forming ones to alter characteristics of the
channel
v. Transmission at different synapses has been argued to be regulated by
different VG Ca++ channels
1. Mammalian NMJ: P type
2. Amphibian/chick: N type

vi. Some synapses seem to have more than 1 type of channel that is regulating
releases (e.g. hippocampal synapses use both P and N type)
vii. Synataxin
1. Associated with release sites
2. T and L type do NOT bind (and thus, dont regulate NT release)
a. L type controls hormone release
viii. Frog NMJ release regulated by N type
1. PHP (blocks R) no effect
2. Agotoxin (blocks P/Q) no effect
3. Conotoxin (blocks N) eliminates EPPs
ix. Mouse NMJ release regulated by P/Q type
1. Agotoxin will eliminate EPPs
x. Hippocampal CA3-CA2 Pyramidal Neurons regulated by P/Q and N
1. DHP (blocks L) no effect
2. Agotoxin (blocks P/Q) reduces EPSP size
3. Conotoxin (N) further reduces EPSP size
xi. Why more than one type?
1. Different gating properties will later the timing of calcium entry
through one channel or another (i.e. differences in voltage
dependence)
2. Different inactivation properties may allow one type to
predominate during certain periods of activity
a. i.e. different response to a train of action potentials
i. N type gradual reduction of calcium current with
each depolarization
ii. P/Q type maintain same amplitude of calcium
current
3. Modulation: one type might be more sensitive to peptide
transmitters in the brain (though GPCRs)
xii. How many calcium channels open under each vesicle during an AP?
1. Experiment: luciferase sensory of Ach in pipette immediately
following single channel opening
a. Detected that sometimes only 1 is needs (i.e. increase in
fluorescence after a single channel opened), although this is
a very low probability
d. Role of Calcium-Activated Potassium Channels
i. These channels bind calcium on the intracellular surface. They open both
when calcium is bound (after entry into the nerve terminal) and when
there is a depolarization
ii. Potassium-Ca++ channels can shape the late phases of AP repolarization
even with single APs, and thus indirectly change transmitter release by
changing voltage pulse (AP) that triggers Ca++ entry
iii. In order to participate at this level, they must be very close to calcium
channels and respond to calcium quickly
1. Block KVG broaden AP from peak and increase peak calcium
and release

a. Opening of these channels is what causes the rapid

repolarization; when they are blocked, the cell is still able
to repolarize because Na++ driving force will cause it to
leak back in, but it will be much slower, thus, broadening
the peak
2. Block KCa broaden AP from half way down and decrease peak
Ca++ and maybe release
a. Returns to good driving force more slowly
b. No after-hyperpolarization

III.

e. LEMS
i. Lambert Eaton Myasthenic Syndrome is an autoimmune disease in
which the body attacks its own Ca++ channels at the active zones of the
NMJ
ii. Clinically related to small cell lung cancer. These tumor cells are like
neuroendocrine cells, and as cancer cells, they are recognized as foreign
by the immune system. Problem comes when the body makes antibodies
to the Ca++ channels on these cells (which in includes P/Q type that
regulates release at the human NMJ).
1. This cross reactivity of antibodies with healthy NMJ Ca++
channels results in the disease
iii. Antibodies reduce the number of functional Ca++ channels present in the
nerve terminal by cross linking them and removing them from the plasma
membrane
iv. Symptoms:
1. Weakness because not all nerve terminals release enough ACh to
cause a muscle action potential
a. When you exercise those muscles, you can temporarily
overcome this weakness due to forced build up of Ca++ in
the terminal
b. If we stimulate these nerves at high frequency, the interval
between activation is short enough that some residual Ca++
is still around to add to the small amount of Ca++ that
comes in with each stimulus. Now NMJs release enough
transmitter to bring the muscle to threshold (e.g. tetanic
potentiation)
v. Clinical Treatment:
1. Treat with blockers of K+ channels (e.g. 3,4-diaminopyridine aka
DAP) prolong the action potential to increase calcium entry,
and thus release
2. New treatments could directly target the Ca++ channel to make
the remaining ones stay open longer
3. We are developing new drugs that act as foot in the door agonists
that directly bind Ca++ channels
4. Using DAP and Ca++ channel agonist will produce a synergistic
effect that restores transmitter release to normal
Mechanisms of Transmitter Release

a. Review
i. Katz quanta of NT = vesicles in nerve terminal
ii. Spontaneous fusion of a vesicle was what led to the measurement of a
mEPP in the postsynaptic cell
iii. Action potential invasion of a nerve terminal led to the fusion of hundreds
of vesicles simultaneously
iv. Are vesicles the substrate for FAST neurotransmitter release? Couldnt
channel function just as well, and faster?
1. Need to fit with quantal nature of release which they do;
channels have characteristic mean open time and conductance
2. Could vesicles only serve as a storage role? Or do they function in
release of slower, non-classical transmitters?
3. Release could be through pores in the membrane (i.e. channels).
ACh is positive at cytoplasmic pH and would flow down its
electrochemical gradient
b. Evidence Against Channels
i. Positive charges of ACh predicts several observations that do NOT occur:
1. Release should alter potential difference across the membrane
(remember, DF = Vm Eq)
2. Changing Vm should alter mEPP size
a. Tested by adding Acetylcholinesterase to presynaptic cell,
but there was no change in mEPP size)
3. ACh release through pores would be concentration dependent
4. There should be a current across the membrane
a. Measured the channel current with patch clamp, but not
current across the membrane
ii. Opening of pores to cytoplasm will create a flow of current through the
pore as ACh moves, but high resolution (double patch clamp)
measurements at frog NMJ shows no gating of current. Thus, a fast gate
or channel model is difficult to support
c. Evidence in Support of Vesicular Theory of Release
i. Direct visualization of these events was achieved 20 years after Katzs
recordings of mEPPs and EPPs
ii. Heuser and Reese used quick-freeze visualization of fusion using a
method called Freeze-Fracture in a resting terminal and in a stimulated
terminal
iii. Experimenters also were able to capture views of an Omega Shape in
TEM thin sections that looked like vesicles fusing with the plasma
membrane
iv. Plasma membrane is a capacitor; the larger the membrane, the larger the
capacitor, and two capacitors in parallel add together2
1. Capacitance measurements: experimenters have detected the
capacitance change associated with large hormone-containing
vesicles fusion events (e.g. mast cells)

2. There are much bigger than typical synaptic vesicles, but now
capacitance change has also been detected form small clear
synaptic vesicle fusion events
v. Spontaneous mEPCs and average capacitance measurement:
1. The capacitance change from a single small clear synaptic vesicle
is so small that the ability to detect it above noise requires the
average of more than 300,000 events
vi. Summary of evidence for vesicles as substrate for release:
1. Freeze fracture dimples near the active zone
2. Transmission EM of section material (omega figures)
3. Capacitance measurements of fusion
d. Mechanisms of Transmitter Release
i. Within the minimal synaptic delay (about 300 to 400 us) following Ca++
entry, the fusion of a vesicle must be triggered. How does this happen?
ii. If Ca++ is a second messenger, how does it signal secretion? How does it
activate a protein-protein cascade? What might Ca++ do inside a nerve
terminal to trigger vesicle fusion?
iii. First thought maybe Ca++ causes phosphorylation of a protein?
1. First guess of Greengard in the early 80s they looked for
proteins that were phosphorylated by nerve stimulation
2. Synpatosomes (pinched off terminals) were added to a test tube
with a solution of 200 mM K+ (to depolarize) and radioactive
phosphate (any phosphorylation will create a radioactive protein)
2D Gel electrophoresis to isolate proteins by charge and size.
Showed about 12 different spots antibodies were made against
these proteins, which were used for selective staining in nerve
terminal and on vesicles
a. One of these proteins showed a staining pattern that was
interesting: on synaptic vesicles synapsin
iv. So action potential activity phosphorylates synapsin, which is found in
the right place (on vesicles). What does it do?
v. Physiological experiment: Squid Giant Synapse Model
1. Purify synapsin incubate in test tube with kinase or phosphatase
2. Inject into nerve terminal no change immediately
vi. Greengard, Llinas et al. injected phosphorylated or dephosphorylated
synapsin into squid nerve terminals, then measured EPPs
1. Dephosphorylated release inhibited
a. Binds up and sequesters vesicles from access to release
sites
2. Phosphorylated No effect
a. Doesnt interact with vesicles
3. CAM Kinase II release increased
a. Phosphorylation of existing synapsin causes the liberation
of more storage vesicles into the ready pool for association
with the docking area. This leads to more release following
each action potential

IV.

vii. How are these result interpreted?

1. Change the state of synapsin phosphorylation regulate the
magnitude of release
2. Synapsin regulates vesicle availability
a. DP sponges up vesicles into storage pool
b. P releases vesicles from the sponge, and thus, more are
available for release
viii. Synapsin is permissive for fusion a slower process than fast Ca++
triggered fusion (i.e. while its involved in transmitter release, it is NOT
the mechanism that is used for Ca++ triggered fusion)
1. Moves vesicles toward or away from active zones based on
whether or not synapsin is phosphorylated
2. Vesicles are not free floating; synapsin, along with actin, heavily
regulates them
Vesicle Trafficking and Fusion
a. Vesicles Trafficking in Nerve Terminal
i. Vesicles have a number membrane proteins that are thought to regulate the
movement of vesicles to release sites. These proteins interact with nerve
terminal cytoskeletal components
ii. The amount of NT that is released with each impulse is dependent on the
number of vesicles that are immediately available for Ca++ triggered
fusion
iii. Favored hypothesis: vesicles are bound within a meshwork of actin
filaments that associate with a protein on vesicles called synapsin-1
iv. Further biochemical and molecular work has revealed that synapsin-1 has
a head domain that binds to phospholipids in the vesicle membrane and
inserts into the hydrophobic region of the membrane. The head domain
also has two high-affinity binding sites for actin. The tail associates with a
specific vesicle membrane protein
1. The key regulatory aspect of this interaction is that the association
of synapsin-1 with vesicles and actin is regulated by
phosphorylation at these sites by cAMP-PK and a specific
calcium-calmodulin-dependent kinase (CaM Kinase)
2. Phosphorylation at site 1 diminishes binding to actin (no effect on
vesicle binding)
3. Phosphorylation at sites 2 and 3 reduces affinity to vesicles and
abolishes binding to actin
v. Although these interactions are predominately thought of in terms of
moving vesicles into the releasing region, pushing the phosphorylation
state of synapsin-1 can have effects on transmitter release
vi. Review
1. DP inhibits release
2. P No effect
3. CaM Kinase II Increases release
vii. This whole process is calcium dependent:

1. The calcium dependent activation of CaM kinase readies

vesicles for secretion following activity of the synapse
a. Higher affinity for Ca++ than the release apparatus, which
it should be, because it is further away from the active
zone, and never experiences the microdomain around the
mouth of the channel
b. It is further away from the calcium channels and needs to
keep feeding vesicles to the exocytosis system
2. Physiological regulation: action potentials at high frequency
increase Ca++ to a level that can temporarily over buffering and
increase calcium concentration in the interior of the nerve
terminal, and this is when we need to liberate more vesicles (not
after only one AP; need large bursts of APs)
b. Vesicle Fusion
i. Fusion of vesicles with the plasma membrane is thought to only take about
100-300 usec. The rest of the 1 msec delay is opening of Ca++ channels,
diffusion of NT, and action on postsynaptic receptors.
ii. In order to achieve this speed, vesicles ready for release must already be
associated with the proteins that will participate in fusion, and are all
waiting for the calcium trigger signal
iii. The breakthrough approach to understanding mechanisms of vesicle
fusion:
1. Came from cell biologists studying non-neuronal cells
2. Organelle movement between compartments (within Golgi, Golgi
to ER, ER to membrane)
3. This Golgi fusion is called constitutive vesicle fusion
a. NO calcium dependent trigger
b. But maybe similar proteins involved
c. Mostly work in yeast easy to manipulate and examine
organelle movement and the effects of mutations that
disrupt this movement. This work led to the identification
of a set of proteins that, when mutated, stopped vesicle
fusion between organelles in yeast cells
4. A series of investigators (Novick, Schekman and Rothman) were
examining constitutive vesicle trafficking in yeast in reconstituted
membrane fusion systems. These findings were used as a key to
look for similar conserved patterns in nerve terminals.
5. The hypothesis: a cell will conserve these vesicle fusion
mechanisms and just add or subtract certain control points
depending on the region of the cell and the specific vesicle fusion
controls needed
a. Many of the proteins involved are related, and this
secretory mechanism appears conserved from yeast to man
b. Similar proteins were identified in the nerve terminal
iv. Basic proteins involved with vesicle fusion: the vesicle docking complex
of proteins and how they might interact to prepare for fusion

V.

1. Vesicles: VAMP, rab-3, synaptotagmin

2. Plasma membrane proteins: SNAP-25, syntaxin, n-sec1, calcium
channels
3. Cytoplasmic components: cytoskeleton (actin), exocyst, NSF,
alpha-SNAP, and ATP
v. The unoccupied vesicle docking area:
1. Syntaxin binds the calcium channel
a. At rest, is bound by n-sec1 (munc-18), inhibiting docking
of vesicles
2. The vesicle is transported to this area by cytoskeletal proteins
vi. Targeting of vesicles to the active zone area:
1. The exocyst interacts with the cytoskeleton to transport vesicles to
the active zone area after they are released by synapsin. Chaperons
the vesicle to the active zone
vii. Tethering of vesicles to the docking area:
1. rab3 (a GTPase) may regulate the alignment of vesicles in the
docking area and may assist in the displacement of n-sec1 from
synataxin
viii. Docking:
1. Synataxin and SNAP25 bind to VAMP to form the CORE
complex, which is an anchor for the rest of the protein-protein
interactions, and also is termed a receptor for NSF and SNAP:
SNARE (Soluble NSF Attachment Receptor)
2. Formation of the CORE complex may displace Ca++ channel
from syntaxin. The calcium channel then may associate with other
proteins in the area (e.g. synaptotagmin)
ix. Priming:
1. The CORE complex binds synaptotagmin and forms a 4-protein
docked complex: VAMP and synaptotagmin on vesicle bind to
synataxin and SNAP25 on the plasma membrane
x. Fusion:
1. This primed rearrangement is on the verge of causing vesicle
fusion, but synaptotagmin provides a calcium-sensitive clamp
that holds it in this tenuous state waiting for Ca++ entry
2. mEPPs may be spontaneous vesicle fusions that get away from
this clamp:
a. Vesicles escape clamp
b. Calcium channels open at RMP
c. RARE release triggered by resting free ionic calcium
xi. Recycling/Recovery Following Fusion:
1. Cytoplasmic NSF/alpha-SNAP break up the CORE complex so
that it can be recycled by joining in the association with VAMP,
SNAP25, and syntaxin, hydrolyzing ATP, and kicking out
synaptotagmin
Mechanisms of Transmitter Release
a. General Scheme

b.

c.

d.

e.

f.

i. Transport (actin)
ii. Targeting (exocyst)
iii. Tethering (rab3)
iv. Docking (core complex)
v. Priming (synaptotagmin clamp)
vi. Fusion (Ca++ releases synaptotagmin clamp)
vii. Recycling of proteins and vesicle membrane (aSNAP/NSF/ATP)
Experimental Support
i. In vitro binding
ii. Peptide fragments that interfere with protein-protein interactions
iii. Mutations/knockouts
iv. Clostridial neurotoxins
In Vitro Binding
i. These experiments led to models, which led to predictions now being
tested at live synapses
ii. Each step in the cascade has been examined using these in vitro binding
assays, but they are not exactly physiological
Peptide Fragments
i. The unoccupied vesicle docking area: syntaxin binds the calcium
channel, which may ensure that the other proteins in the complex are
either close by or can bind the calcium channel at a later step
ii. Experiment: inject peptide fragments that mimic the calcium channel
binding site to syntaxin uncouples calcium channel from syntaxin
association decreases release and shifts calcium/release relationship
to a linear one
iii. Can be overcome by increasing extracellular calcium
iv. Thus the resting docking site may ensure the important players
Mutations and Knockouts
i. In drosophila, mutate n-sec1 (unc-18) severe synaptic paralysis, lethal
mutants
1. A complete block-of-release phenotype tells us that the protein was
essential for transmitter release, but it does not tell us what it was
used for
ii. Rab3 knockout mice show normal release initially, but depression with
continued stimulation important for maintaining a steady stream of
reserve vesicles for docking
iii. N-sec1 is required; Rab3 assists, but not necessarily needed
Tetanus and Botulism
i. TeTx 1 toxin
ii. BoNT 8 different toxins (A-H)
iii. All 9 toxins have 2 chains
1. Heavy (B) binds to receptors on nerve terminals to get toxins
across the membrane
2. Light (A) acts as a peptidase to cleave fusion-associated proteins
3. Just A chains cannot get in the terminal; need B chains to bring it
in!

iv. Targets
1. TeTx VAMP
2. Bot B, D, F, G VAMP
3. Bot A, C, E SNAP 25
4. Bot C Syntaxin
v. Environmental Distrubution
1. Clostridium tetani exists as spores that will germinate in anaerobic
environments
2. Clostridium botulinum contaminates food in anaerobic
environments
vi. Clinical problems
1. Tetanus attacks CNS; taken up by NMJ and retrogradely
transported back up to the cell body, then moving transsynaptically to neurons that synapse on motor neurons
a. Spastic paralysis
2. Botulinum directly attacks the NMJ synapse; usually ingested;
attacks PNS; no retrograde transport, acts locally
a. Muscular weakness or paralysis
vii. Treatment
1. Neutralize with antitoxin ABS
2. Prevent with vaccines
g. Triggered Fusion
i. Ca++ rise of 10-100 uM causes fusion to begin
ii. There are several proteins identified in nerve terminals that bind Ca++;
what is the evidence that synaptotagmin is the sensor that triggers APevoked release?
1. Synaptotagmin has two C2 domains (which in PKC bind Ca++)
a. First domain binds Ca++ in the 100-200 uM range (lower
affinity) fits with expectations of calcium concentration
next to an open channel
b. Following the solution of the 3D structures of these
domains, it appears that:
i. C2A binds 3 Ca++
ii. C2B binds 2 Ca++
c. Calcium binding leads to a change in molecular interactions
may lead to the non-linear relationship between calcium
binding and transmitter release
d. Calcium binding may decrease association of C2 domains
with other vesicle membrane proteins and cause them to
dimerize with one another
e. Calcium alters binding of this protein to the plasma
membrane by neutralizing charge, causing it to penetrate
the plasma membrane.
2. Synaptotagmin associates with the calcium channel after the
vesicle docks; it is close to Ca++ entry

VI.

3. Knockout gene experiments in mice have shown that

synaptotagmin mutants lose the fast, synchronous component of
release, and instead have a slow, asynchronous release following
AP
a. Thus, it is possible that synaptotagmin is the low affinity
Ca++ receptor that is the primary sensory for release
b. However, there may be another way transmitter release can
be triggered in a less efficient way in the absence of
synaptotagmin
4. Drosophila Mutations and Knockouts
a. Change 1 amino acid (mutation AD3) shift Ca++
sensitivity; NT release controlled at same order, just need to
increase [Ca++]
b. Cut off one C2 domain (mutation AD1) change the 4th
order relationship to 1st or 2nd order
c. Predicts that knockouts would have no regulated calciumtriggered secretion and that spontaneous release would
increase
h. Reversal of the SNARE Protein Interactions
i. Cytoplasmic alpha-SNAP binds to syntaxin
1. Appears to be required for exocytosis if you introduce a peptide
fragment of this molecule into terminal, you will block proteinprotein interactions, and thus release
a. Needed to recycle vesicles
ii. NSF binds alpha-SNAP causing ATP hydrolysis
1. Dissociates the SNARE-CORE complex
2. ATPase activity is used for the energy of this rearrangement
required for recovery
3. Peptides against NSF portions that mediate ATP hydrolysis
compete for ATP binding and prevent hydrolysis
a. 80% block of release, but only after use
b. Release tends to go down as CORE proteins cant uncoil
c. Will initially work, but vesicles cant be replaced
Endocytosis
a. Proving that Vesicle Membrane is Retrieved
i. EM (quick freeze section or freeze fracture)
1. Evidence of membrane pinching back in, a little away from sites of
fusion (however, slower than exocytosis)
2. In lamprey, where vesicles cluster in obvious clouds around the
active zone, it is obvious that the regions for endocytosis are NOT
the same as regions for exocytosis
ii. Dyes
1. Incubate synapse with dye or colored marker
a. Stimulate in HRP (1Hz, 15 min, wait 1hr) stains vesicles
b. Stim same synapse again in absence of HRP no staining

iii.

iv.

v.
vi.

2. Vesicles recycle. Similar experiments have been done with

fluorescent dyes in living preparations to directly visualize staining
and de-staining
Radioactive ACh Precursors
1. Stimulate a terminal with radioactive ACh in synapse
Radioactive ACh taken up stim again Stain back in synapse
(terminal de-stained)
2. Suggests that vesicles go back to the ready pool
a. The storage pool is held there by dephosphorylated
synapsin. Newly recycled vesicles are NOT caught in this
meshwork and can move to an active zone area
Retrieval of the Right Piece of Membrane
1. Experiment: label vesicle membranes with antibody to
synaptophysin in the bath.
a. After stimulation of exocytosis: stained nerve terminal
b. After endocytosis: cant stain anymore
2. Membrane retrieval needs to be done locally and fast to keep up
with demand (endocytosis occurs in 1-5 seconds)
3. The vesicle is recycled, refilled with transmitter, and ready for
secretion again in about 30 to 60 seconds
4. Synaptic vesicles have a half-life of about 2 to 3 days
5. Recycling appears to be a receptor-mediated event; that is, there
appears to be something on the vesicle membrane that is
recognized so that some apparatus that pulls it knows where to
grab
6. Internal budding of membranes often involves the assembly of a
cage-like coating called clathrin-coated pits
7. The mechanisms of endocytosis appear to involve these coated
pits and Ca++
a. Isolated coated vesicles from brains are mostly synaptic
vesicles
b. Number of coated vesicles in nerve terminal increase more
than ten times after stimulation
8. Clathrin needs a guide, Adapter Protein 2, to tell it where to form
9. AP2 binds to pieces of membrane that are vesicle membrane by
binding synaptotagmin and the first C2 domain
10. After these areas of membrane are identified and caged in clathrin,
they need to be pinched into the cell (budding)
11. This process probably mediated by another protein called dynamin
12. Dynamin is a GTPase that can form rings (GTP gamma is a form
of GTP that the experimenter can add to prevent GTP cleavage
rings cant complete the pinch of the budding vesicle)
Mutation of drosophila dynamin, which functions in clathrin-mediated
endocytosis, depletes synaptic vesicles following stimulation
Endocytosis is blocked by removing extracellular calcium, although the
function of calcium is likely to result from either general decrease in

intracellular calcium, or elimination of the intracellular calcium increase

that occurs with stimulation
vii. Ca++ Dependent Recycling
1. Clathrin coated vesicle membrane, assisted by AP2 binding to
synaptotagmin (which is calcium dependent)
2. Dynamin is dephosphorylated in a calcium-dependent manner, and
this could mediate or stimulate endocytosis
b. Other Related Issues
i. Kiss and Run
1. Transient opening and closing of a docked vesicle, which is then
refilled
2. Hard to get good evidence of this
ii. Bulk Endocytosis
1. Also may occur, but only after very intense stimulation
2. Probably not physiological
iii. Dense-Core Vesicles
1. Dont recycle
2. Peptide and hormone release is far from larger dense core
vesicles (full of proteins). These cannot be recycled because cant
reload them with proteins or peptides at nerve terminals need to
make these proteins in the ER and package in the Golgi apparatus