W3 Cholinergic Neurotransmission (Dustin) Flashcards
What are the 4 main locations for cholinergic neurons?
- Neuromuscular junction (motor neurons)
- Autonomic preganglionic fibers (both sympathetic and parasympathetic)
- Parasympathetic postganglionic fibers
- Central nervous system
Where is acetylcholine synthesized?
In the cytosol, afterwards it is stored in vesicles
For this reaction, name the substrates and products
What’s the enzyme?
Where is the enzyme (cell type and intracellular compartment)?
Substrates: choline + acetyl-CoA
Products: Acetylcholine + CoA-SH
Enzyme: choline acetyltransferase (ChAT)
Location: cytosol of cholinergic axon terminal
What triggers acetylcholine release from the axon terminal?
Calcium signal
Action potential -> depolarized axon terminal -> calcium entry via voltage-gated calcium channels -> exocytosis of vesicles
What is the name of the enzyme that hydrolyzes acetycholine in the cholinergic synapse?
What kind of molecules exert an irreversible inhibition of this enzyme?
What is the underlying effect of the irreversible inhibition of the enzyme by those molecules?
Acetylcholinesterase
Organophosphates, i.e. DFP, Sarin gas. Makes pesticides and chemical weapons.
Phosphorylates a serine group of Ach-esterase, making it unable to function and this cannot be undone (at least in the CNS, the antidote for this only works in the periphery). The action of acetylcholine on nerve terminals becomes continuous, so both constant muscarinic and nicotinic effects.
Where does most of the acetyl-CoA used in formation of acetylcholine come from?
Glucose metabolism, only in long-term starvation will it come from other sources
What is most capable of limiting acetylcholine synthesis?
What is NOT rate-limiting in ACh synthesis?
Choline uptake, but still not likely. Has high affinity to its transporter that’s specific for cholinergic neurons
There is plenty of acetyl-CoA and choline acetyltransferase (ChAT), so these are not rate-limiting.
What inhibits choline uptake in cholinergic neurons?
Hemicholinium
What are 6 steps in the cycle of vesicles going through uptake of their neurotransmitter, release, and back again?
- Uptake of NT into vesicle
- Formation of cluster of vesicles - reserve pool
- Docking of vesicles into the active zone: there will not be actual physical contact between the vesicle and plasma membrane
- Priming- vesicles become ready for calcium-induced fusion
- Calcium signal -> fusion to lipid membrane
- Vesicle is recycled
Sometimes they can touch the membrane without NT release in either the “kiss and stay” or “kiss and run” way
Where are SNAPs? Where are VAMPs?
What do these acronyms stand for?
SNAPs are in the plasma membrane, VAMPs are in the vesicle membrane
SNAP = synaptosomal-associated protein
VAMP = vesicle-associated membrane protein
What happens between VAMPs and SNAP-25 to prepare a vesicle for calcium stimulus (“priming”)?
The synaptobrevin part of VAMP forms a SNARE complex with the syntaxin 1 and SNAP-25 in the plasma membrane, which forms a coil and further supercoil, which provides energy for fusion of the vesicle and the plasma membrane
The SNARE motif draws the membranes closer together “like a zipper”
What is synaptotagmin?
This is a calcium sensor in the plasma membrane of presynaptic axon terminals
The synaptotagmin C2 domain originates in the synaptic vesicle and then is placed into the plasma membrane cytosolic surface after a vesicle has undergone “priming” and is ready for a calcium signal to exocytose acetylcholine
Calcium will then trigger fusion, and acetycholine will then be released
What toxin is specific for the SNARE motif proteins used for the cholinergic neurons?
How does it work?
Botulinum toxins
Have Zinc-dependent endoprotease activity that cuts SNARE motifs at different sites, inhibiting exocytosis of Ach by either increasing or decreasing the stability of SNARE complexes
What is the main difference between nicotinic and muscarinic acetycholine receptors?
What are their major inhibitors?
Nicotinic receptors are ligand-gated receptors
Muscarinic receptors are G-protein coupled receptors (GPCR’s)
Nicotinic is inhibited by curare, muscarinic by atropine
Which type of GPCR is found in the M1 muscarinic acetycholine receptor? What are those GPCR effects (briefly, what’s the cascade)?
M1 = Gq
-> phospholipase C -> PIP2 -> IP3 -> Calcium signal
Which type of GPCR is the M2 receptor?
Where in the body is it found, importantly?
What are its effects?
M2 = Gi
Found in the heart
The G-protein alpha subunit activates potassium channels, which leads to hyperpolarization of the SA/AV nodes which are then harder to depolarize, resulting in decreased chronotropy / lower heart rate
What type of GPCR are M3 receptors?
Where are they found, importantly?
What is their function?
M3 = Gq
Found in the gastrointestinal tract
Participate in smooth muscle contraction and stimulation of gastrointestinal secretions
How many subunits make up the nicotinic acetylcholine receptor?
5 subunits total (pentamer)
2 identical alpha subunits
then there is 1 each of beta, gamma, delta
What is the direct effect of nicotinic acetylcholine receptors?
Sodium and potassium become permeable, resulting in membrane depolarization
In muscle, this leads to calcium being released from the sarcoplasmic reticulum
What toxin is specific for only the muscular nicotinic acetylcholine receptors?
alpha-bungarotoxin (a snake venom)
Leads to paralysis
What is an autoimmune attack of nicotinic receptors called?
What occurs?
What is the first symptom of this?
Myasthenia gravis
The number of receptors is decreased because their degradation is accelerated, plus existing receptors are blocked by antibodies
First symptom of this is fatigue, which progressively gets worse
How specifically is calcium released from the sarcoplasmic reticulum after nicotinic acetylcholine receptors are activated in skeletal muscle?
L-type calcium channels (DHP in particular) react to depolarization of the plasma membrane, but do not function as calcium channels themselves. They have close physical contact with Ryanodine receptors, which are in the SR and allow calcium to be released into the cytosol
Where in the body is the ryanodine (RYR) type 1 receptor?
Where is the RYR2?
RYR1: skeletal muscle
RYR2: heart muscle
What condition results from mutation of the RYR1 receptor?
Malignant hyperthermia
Calcium continuously leaks from the SR, causing muscle rigidity
Other effects: accelerated metabolism (because some citric acid cycle enzymes are stimulated by calcium: pyruvate dehydrogenase, alphaketoglutarate dehydrogenase, and isocitrate dehydrogenase)
Which neurotransmitters are inactivated enzymatically?
Only acetylcholine is
What is the significance of “pseudocholinesterases” - cholinesterases that are in the blood plasma, and not in the cholinergic nerve terminal?
They hydrolyze drugs that are used as short-lasting muscle relaxants. If there is a mutation in these enzymes, it would not be known until the person undergoes anesthesia, and may take much longer than normal for anesthetics to lose their effect
Example of one such drug used in anesthesia = suxamethonium
What is used to test for myasthenia gravis?
What about treatment?
Why?
To test: a short-lasting, reversible cholinesterase inhibitor
To treat: a long-lasting reversible cholinesterase inhibitor
Reasoning: keeps acetylcholine around the nerve terminal for longer, increasing the probability that it will bind to the receptor
Why might cholinesterase inhibitors be used to treat glaucoma?
Glaucoma = increased pressure in the eye
Acetylcholinesterase in the form of eye drops increases the constriction of the pupil (parasympathetic stimulation), which can decrease that pressure
What is the major family of irreversible cholinesterase inhibitors?
What are some examples?
How do they work?
Organophosphates
as in DFP (pesticide), Sarin gas
They are lipophilic, and easily penetrate membranes. Once in the system, they irreversibly phosphorylate the serine group of acetylcholinesterase, which makes it non-functional. Acetylcholine stays in the nerve terminal -> extreme cholinergic effects -> depression of the system.
Atropine is the emergency treatment but does not fix the enzyme. There are some partialy effective “antidotes” but they only work for peripheral acetylcholinesterase, while the organophosphates also act on the CNS
What is the normal plasma choline concentration?
What is “low affinity” choline uptake’s Km and location?
What about “high affinity”?
Normal plasma concentration: 10 micromolar
- Low affinity: Km > 10 micromolar + in all tissues
- High affinity: Km = 1-5 micromolar + in only cholinergic neurons
(slide said normal was 10 millimolar, but that didnt make sense for the “low affinity” uptake having micromolar Km… looked it up and normal is micromolar)
