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53 Cards in this Set

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What takes most time in postsynaptic delay
bring Ca into the Ca channel , takes 1/2 millisecond
What role does Ca play
Ca is the trigger for chemical activation
What experiments did Katz do to determine role of Ca
if Ca concentration is 0, we predict no transmitter release - no EPP recorded
added Ca of ionophoretic pipette at various times
after stimulation - no EPP
before stimulation - EPP
What did Katz conclude
Ca has to be there before stimulation (extracellular)
What happens when you add Mg
Mg is a competitive ion, also divalent, will block stimulation (block Ca channel), another blocker - Cd
What other experiments could be done
bypass channel to dleiver Ca
Ionophore
a molecule that will insert into membrane and create artificial Ca channel
Ca liposome
doesnt add pore to membrane, lipid contianing Ca bundles
Caged Ca
when you inject free Ca, nothing happens. Molecule bound Ca until hit by light from laser and then loses affinity for Ca and releases it, will get EPP, mimicks how Ca actually gets in, occurs w/out stimulation of nerve or using voltage gated channels.
What is the result of the alternate experiments
all 3 result in NT release, so AP not required, just the way to get Ca in.
Ca homeostasis
Ca will not make it to active zone inside of cell. 100 nM inside, outside 2 mM. Huge difference between inside and outside. Mechanisms maintain low Ca inside - cant use as signaling molecule if not low inside
Ca/Mg ATPase
pump uses ATP as energy supply to pump Ca back out of the cell. Mg needed as cofactor for ATP binding. 1 ATP = 1 Ca out. Pump activated by binding of Ca
Na/Ca pump
no need for ATP, uses Na only when concentration is in micro level (very big)
1 Ca - every 3 Na in, doesnt contribute to AP
intracellular stores
store in ER and mitochondria, liberated by 2nd messenger (Ca, IP3). Organelles sponge up big influx of Ca
Proteins
Ca binding proteins work as buffers
Name 2 exogneous buffers
EGTA and BAPTA
EGTA
relatively slow, high affinity
BAPTA
fast, high affinity. Blocks transmitter release, can make it a fluorescent molecule to trace where Ca was bound. To figure out concentration need to combine caged Ca with fluorescent BAPTA
Tetanic potentiation
many AP in burst, postsynaptic response summates (accumulating Ca since no time for buffering by homeostasis)
Post tetanic potentiation
After giving tetanus - residual Ca
LEMS Lamber Eaton Myasthenic Syndrome
autoimmune attack on NMJ's.Patients with small cell lung cancer (neuroendocrine) - similar to neuron - releasing peptides like NT, have Ca channels etc. Immune system making antibodies to proteins - nerve terminal ends up with fewer presynaptic channels then usual. Bind to active zones of Ca channels.50 % of Ca channels lost - get smaller EPP. fewer Ca entry sites, fewer quanta release, so muscle cells do not reach threshold - causes weakness. Exercising helps since Ca is building in the nerve terminal due to residual Ca
Repetitive Neural Stimulation Test
connect thumb muscle to electrode, if response gets progressively larger, then suspect LEMS
How would you treat LEMS
block K channel, will make AP longer and will get bigger Ca current
2 fold change in Ca concentration would lead to ... change in response
2 ^ 4 , or 16 fold change
How can you determine type of Ca channel
by blocking with natural toxins
What type of Ca channel is typical for mammalians
P/Q
What toxin does P/Q respond to
omega AGA IV toxin
Why do we have multiple Ca channel types
Each Ca channel unique, Ca channels do not inactivate as well as Na channels, modulation of channel directly by phosphorylation leads to greater plasticity
Ca activated K channel
hybrid of voltage gated channel and ligand gated channel, need Ca entering + depolarization, responsible for AHP, if block then no AHP, doesnt return to good driving force, and less Ca entry
What is the purpose of Ca activated K channels
abrupt repolarization
How do we know we dont have channels instead of vesicles
do whole cell patch clamp, if channel - should see current, Vm didnt change so its not a channel
Freeze fracture technique
Heuser Reese, instead of fixing tissue, freeze it, bilayer splits open, looked at presynaptic structure - showed vesicles are fusing
Name 3 evidences for vesicle mediated release
freeze fracture, section frozen material, and capacitance change
WHat happens when dephosphorylated synapsin enters
release decreased
What happens when inject phosphorylated synapsin
nothing no change
What happens when inject CAM kinase II
increased release, phosphorylation decreases affinity of synapsin for vesicles and actin
Name 5 cytoplasmic components in fusion
1. actin= cytosceleton
2.NSF
3.ATP
4.SNAP
5 Exocyst
What is the first step of fusion
liberation of vesicle from actin by phosphorylation in storage pool and actin transporting vesicle to release pool
What is the function of RAB 3
chaperone protein, removes nsec from syntaxin, allowing formation of core complex
What is core complex
VAMP + syntaxin + SNAP 25, same as SNARE proteins, coil together to form " coiled-coil"
Name proteins that are located on vesicle
VAMP, synaptotagmin, RAB 3
Name proteins on membrane
Ca channel, syntaxin, SNAP 25
Experimental support for the model
in vitro binding, competetive peptides, mutations(KO) and toxins
In vitro binding
done in test tube, so may not be accurate in body. You either homogenize brain to free proteins in solution or cloned proteins.
1. put syntaxin+ vamp+ snap in test tube
2. add antibody to syntaxin
3. centrifuge
4. run on gel
If they are connected, antibodies for one will bring others down as well
What happens if you start with nsf alpha snap ATP and add syntaxin, snap 25 and VAMP
core complex doesnt form
What is the disadvantgae of in vitro binding
doesnt tell about sequence, just what associates with what under various conditions
Competetive peptides
interfere with normal binding, stim - EPP normal, stim with peptides NT release decreases, microdimains further from vesicles
What toxins cut core complex proteins
tetanus and botulism, they abolish transmitter release - get no EPP
What happens when you knock out RAB 3 gene
initially looks normal, with time diminishes, so RAB 3 is helpful but not essential
Why synaptotagmin considered to be Ca sensor
1. It has binding sites for Ca - has C2 domains at repeats A and B, binds 4-5 Ca ions, not 1:1 ration but 4 fold
2. also Ca dependent, binds with low affinity, i.e need large amount of Ca to bind - microdomains
What did knock out synaptotagmin show
that it is responsible for SYNCHRONIZED release
What happens when you remove 1 C2 domain of synaptotagmin
release changes from 4 fold to 2 fold
What happens when you change one amino acid in a synaptotagmin sequence
remains 4 fold, but shifts to right