exam 2 cards Flashcards
what steps occur in vesicle secretion?
Action potential, nerve terminal depolarization, activation of voltage gated calcium channels, calcium enters down a strong gradient , calcium triggers transmitter release, exocytosis of neurotransmitter, transmitter crosses synaptic cleft, transmitter binds to receptor, postsynaptic conductance change, action potential
what is synaptic delay?
time between a presynaptic AP and a postsynaptic effect (1 msec)
what step in neurotransmission takes the most time?
activation and entry of calcium
What happens if you decrease calcium in the cell?
you will decrease the amount of NT secreted
What part of the AP is calcium entry?
the falling phase of the AP
What is the difference in [Ca++] intracellularly vs extracellularly?
Calcium is typically very high outside (2 mM) and very low inside (10-100 nM). This is 5 orders of magnitude greater on the outside of the cell versus the inside of the cell. This allows calcium to be a signaling ion.
Katz experiment: when you remove calcium from the bath, what happens to AP and vesicle release of the neurons?
there is no longer vesicle release, although AP and postsynaptic sensitivity are the same.
What happens when you add an ionic blocker for calcium (Mg++, Cd++)?
These compete with calcium and will block the release of calcium in the cell.
What happens when you include calcium ionophores/ liposomes?
first ionophores are molecules that package another molecule so that they can go across the plasma membrane. a liposome works by a similar method, the molecule is just packaged in lipids so it can transport across the membrane.
This allows molecules to be directly transported inside the cell. In this case a calcium ionophore or a liposome will cause vesicle release.
what happens when you load a terminal with caged calcium?
First… caged calcium is calcium that is trapped until it is released by a light signal.
Once the calcium is liberated (but not before) transmitter release occurs.
Calcium is necessary for….
release of neurotransmitter
Where in the cell is the concentration of calcium most important? How do we make sure it gets there?
[Ca++] is most important around the terminal.
an easy way to make sure it is used there is through a calcium ionophore.
What are the major calcium transporters in the cell?
Ca- Mg- ATPase, Na-Ca exchanger. These are important to maintain a relatively low level of intracellular calcium
What does Ca-Mg- ATPase do?
This transporter uses ATP to transport calcium out of the cell (against concentration gradient). It has a very high affinity for calcium.
This requires Mg as a cofactor for ATP binding
This mechanism results in one calcium transported out of the cell for every ATP hydrolyzed.
What does Na-Ca exchanger do?
This is activated by intracellular calcium, but has a much lower affinity than Ca-Mg- ATPase does.
this process is driven by the electrochemical gradient for Na
One calcium is extruded for every 3 Na+ ions entering the cell.
this is also an electrogenic pump that will depolarize the cell.
There is a low concentration of the Na-Ca exchanger in the cell, so it does not have a robust effect.
Calcium buffers: what are they and what is the effect?
These are free floating proteins that bind to calcium and lower the [Ca++] in the cell.
Typically these buffers include the ER, mitochondria, and proteins.
Explain how the two types of buffers (EGTA and BAPTA) work and how they
EGTA has a high affinity for calcium and a low speed of binding, BAPTA has a low affinity for calcium and a fast speed for binding.
EGTA will have little to no effect on vesicle release because it isn’t fast enough.
BAPTA will block release because it binds more quickly than the vesicle can be released.
You need a fast buffer to grab calcium after it enter the nerve terminal but before it binds to the secretory apparatus… this means that calcium entry and release must be very close to one another.
What concentration of calcium do we need to get transmitter release?
10-100um intracellularly.
what is a microdomain?
these are the calcium right under the calcium channel that trigger NT release.
What is short term plasticity? What increases it?
This is a balance between residual calcium effects that increase release and the depletion of vesicles.
This means that some calcium stays in the cell from the last AP, and then more rushes in form the next one. This increases [Ca++] overall and then increases the amount of NT released.
paired pulse facilitation
when two stimuli are delivered in rapid succession, the second stimuli evokes a larger response than the first.
tetanic potentiation
during a train of multiple stimuli at high frequency, there is a gradual increase in the amount of NT released.
post-tetanic potentiation
after a train of stimuli has ended, there is an increase in the amount of NT released with a single stimuli at a low frequency for some time.
lambert eaton myasthenic syndrome
theis is an autoimmune disease where the body attacks its own calcium channels at the active zones of the NMJ
This results from cancer in another part of the body. The body creates antibodies that attack the calcium channels of tumor cells. the antibodies for this cause LEMS because the antibodies attack the similar calcium channels in the calcium channels of the NMJ.
These antibodies reduce the number of functional calcium channels in the nerve terminal by removing them from the plasma membrane.
This makes people feel weak because not enough ACh is being released.
Treatment for lambert eaton syndrome
treat with potassium channel blockers. This will prolong the AP to increase calcium entry into the cell, and will therefore increase NT release.
Possible new treatments for LEMS
directly target the calcium channels to make the remaining channels stay open longer, drugs that act as agonists on calcium channels.
equation for describing relationship between calcium concentration vs. measure of voltage.
(multiple of the concentration change of calcium)^3 or 4 = multiple in the change in voltage.
example. .15-.3 change in calcium concentration (2 fold), equals a post synaptic change from .25 mV to 4 mV (16 fold)
2^4= 16.
This means that a small change in the calcium concentration of a synapse will cause a large change in the synaptic measure of release
What does the non-linear relationship of NT release to Ca concentration say about the mechanism?
It says that there must be multiple binding sites that bind in a cooperative manner for release to occur. This means that either one protein with multiple sites bind calcium, or multiple proteins, or something in between.
if it was just one binding site, the relationship would be linear.
What are the types of calcium channels that regulate NT release?
P- type and N-type. Some cells use a combination of the two.
Why would you use more than one type of calcium channel at the same synapse/
this allows for plasticity. different gating proterits will affect the timing of calcium entry.
different inactivation properties may allow one type to predominate during certain periods/ types of activity.
evidence against vesicles only having a storage role
the + charge of ACh predicts that release of ACh via a channel should alter the membrane potential
Changing the membrane potential would change the mEPP size
ACh release would have to be concentration dependent
Evidence in support of vesicular theory of release
direct visualization in freeze-fracture in a resting termina versus a stimulated terminal where vesicles are lined up against the pre and postsynaptic ends
This kind of image could not be achieved with chemical fixatives because the fixatives would kill the tissue.
what do capacitance experiments tell us about the nature of vesicles?
The capacitance change from vesicle release is negligible.
This means that the current doesnt change as predicted by release of NT without a vesicle.
how did researchers come up with the protein that calcium activates?
they incubated the nerve terminals in 32P.
Then they stimulated the terminal and isolated all phosphorylated proteins (12).
They then made antibodies against each type of protein to stain the cells.
they found one that bound to vesicles. This was “synapsin”
they isolated synapsin to look at structure, function.
difference between a phosphotase and a kinase?
a kinase phosphorylates
a phosphotase dephosphorylates.
what happens when you inject dephosphorylated form of synapsin into the giant squid synapse?
decreased NT release, the synapsin binds up and sequesters vesicles.
what happens when you inject phosphorylated form of synapsin into the giant squid synapse?
no effect, the phosphorylated form will not interact with vesicles.
what happens when you inject Cam kinase (a phosphorylating agent) to the giant squid synapse?
transmitter release is increased.
This causes phosphorylation of existing synapsin from docking areas
So what does synapsin do?
it regulates the avaliability of vesicles to participate in the release process.
This process is permissive for fusion, not fast calcium-triggered fusion.
What does dephosphorylation do to the availability of vesicles?
it makes vesicles unavaliable
What does phosphorylation do to the availability of vesicles?
it makes vesicles avaliable
What are the tow parts of calcium dependent regulation of transmitter release?
vesicle trafficking and vesicle fusion
How are vesicles bound?
through a meshwork of actin filaments that are bound to synapsin
how does synapsin-1 bind to vesicles?
synapsin-1 has a head domanin that binds to phospholipids in the vesicle membrane and inserts the hydrophobic region of the membrane
how does synapsin-1 bind to actin?
it has two high affinity sites for actin in the head region
synapsin is bound to actin in the —— state
dephosphorylated state
phosphorylation at site one in the synapsin has what effect?
It diminishes binding to actin
phosphorylation at site two and three in the synapsin has what effect?
reduces affinity of synapsin to vesicles and abolishes binding to actin
What does CaM kinase do (calcium calmodulin-dependent kinase)?
It phophorylates synapsin, but is dependent on Ca++ concentration in the cell.
what happens when you inject dephosphorylated synapsin into the cell?
it inhibits release of vesicles because it binds actin to vesicles and synapsin
what happens when you inject phosphorylated synapsin into the cell?
no effect
what happens when you inject CAM kinase into the cell?
increase release because it phosphorylates synapsin.
what is the hypothesis of vesicle fusion throughout the body?
It is believed that all vesicles are made in a similar manner, the only thing that is different is the control points in the mechanism that are necessary for different parts of the cell.
what are the six cytoplasmic components involved in vesicle docking?
cytoskeleton, the exocyst, NSF, SNAP, ATP
What are the 4 plasma membrane proteins involved in vesicle docking?
SNAP-25, syntaxin, n-sec1, as well as the calcium channel
what are the steps of vesicle fusion?
1) syntaxin (which is associated with n-sec1) binds to the calcium channel
2) the vesicle is transported to the area by cytoskeletal proteins
3) the exocyst transports vesicles to the active zone area after they are released by synapsin
4) the vesicle is tethered to the docking area by Rab3 protein (a GTPase). This is thought to displace nsec1 from syntaxin as well as ready other machinery
PRIMING
5) syntaxin and SNAP 25 bind VAMP (on the vesicle) to form the core complex
6) VAMP and synaptotagmin on the vesicle bind to syntaxin & SNAP-25 on the plasma membrane
FUSION
7) synaptotagmin provides a calcium sensitive clamp that holds the vesicle awaiting calcium entry
RECYCLING/RECOVERY
8) the CORE complex dissociates, and is recycled by joining in association with VAMP
what is an exocyst?
which is a complex of proteins that interact with the cytoskeleton, these are chaperon proteins that aid movement of vesicles from synapsin storage to the active zone area
what does rab 3 do?
the vesicle is tethered to the docking area by Rab3 protein (a GTPase). This is thought to displace nsec1 from syntaxin as well as ready other machinery
what is in a V-snare?
VAMP and synaptotagmin
What is in a T-snare?
syntaxin and snap 25
simiplified version of vesicle release
- transport (actin)
- Targeting (exocyst)
- Tethering (rab)
- Docking (core complex)
- Priming (synaptotagmin clamp)
- fusion (calcium releases synaptotagmin clamp)
what would happen if you bound a protein to syntaxin that interfered with Ca++ receptor binding?
it would decrease release and make [Ca++]/release relationship very similar
this is because this step ensures that calcium channels are nearby.
what happens if a nsec1 gene is mutated
It will interfere with formation of the core complex, if nsec1 doesnt dissociate, the vesicles can’t dock.
what is the purpose of rab3?
to displace nsec1
it is important for creating reserve vesicles for subsequent docking
what is the target of tetanus?
VAMP
What are the targets of Botulism toxin?
snap 25, syntaxin, and VAMP
what are the symptoms of tetanus? how does it act?
spastic paralysis, body exhaustion, respiratory failure. It starts in the NMJ and then transports to the CNS
What are the symptoms of botulinium poisoning? How does botulinium poisoning act?
directly attack NMJ synapse, usually ingested, gets in blood stream and teds to focus attack on the PNS
how do we treat botulism and tetanus?
we use the anti toxin Abs and vaccines
What is the current belief on calcium triggered fusion?
Calcium alters the binding of synaptotagmin, which causes synaptotagmin to puncture the membrane.
this has been confirmed by finding 3 different calcium channels on synaptotagmin as well as doing knockout experiments for synaptotagmin.
what happens when you knock out one of the calcium receptor domains on synaptotagmin?
It depends on the domain. For mutation of the 2nd domain slightly, it is still 4th order, but needs more calcium.
for complete mutation of the second domain, the order of the relationship shifts from 4th –> second order relationship
What does alpha snap do?
It introduces a peptide fragment into the terminal and blocks protein/protein interactions. this blocks release of vesicles.
What does NSF do?
It binds alpha- SNAP causing ATP hydrolysis, and it dissociates the SNARE CORE complex
would would happen if there was only exocytosis and not endocytosis?
The plasma membrane would just constantly increase in area.
What happens when you stimulate a neuron at high frequency for a long time?
you will start to see increase in PM area, and depletion of vesicles in the membrane
This suggests that the membrane has to be retrieved in some way
What happens when you stimulate a cell with a single stimuli at low frequency?
nothing really changes about the plasma membrane
what are the results when you stimulate a cell in HRP infuse medium?
The vesicles will be stained with HRP, but nothing else
If you stimulate without HRP, the vesicles are also not stained.
Do recycled vesicles go immediately to the storage pool or the readily useable pool?
they go immediately to the readily useable pool.
This happens because the dephosphorylated synapsin is most likely already binding maximum vesicles. And because these are occupied, it would be hard to bind more.
what is the half-life of a synaptic vesicle?
2-3 days
How do you tap into the storage pool of vesicles?
stimulate the cell mildly with low electricity
What is clatharin?
The coating that lines a vesicle during endocytosis
what is the guide for formation of clathrin?
AP2 (adapter protein 2)
What protein pinches off the vesicle?
Dynamin
What is dynamin?
a GTPase that can form rings to pinch off a budding vesicle.
what happens with a mutation in dynamin?
it depletes synaptic vesicles following stimulation. It can also make long, non-functional vesicles by incorrect dynamin binding.
How is clatharin formation regulated by calcium?
Clathrin is assisted by ap2 binding to synaptotagmin, which has Ca++ binding sites.
Dynamin is dephosphorylated by a Ca++ dependent manner. It is phosphorylated to decrease its activity, and can be dephosphorylated by a calcium-dependent phosphatase to increase activity)
Both of these mechanisms could contribute to calcium dependent exocytosis.
which has more activity, phosphorylated or dephosphorylated dynamin?
dephosphorylated
What are the three ideas of how vesicle endocytosis works?
1- Kiss and run: the vesicle is constantly refilling and dumping, stays in one place.
2- clathrin method
3- bulk endocytosis: large amounts are transported in, then broken up later into vesicles.
