W3 Cholinergic Neurotransmission (Dustin)

Question 1

What are the 4 main locations for cholinergic neurons?

Answer 1

1. Neuromuscular junction (motor neurons)
2. Autonomic preganglionic fibers (both sympathetic and parasympathetic)
3. Parasympathetic postganglionic fibers
4. Central nervous system

Question 2

Where is acetylcholine synthesized?

Answer 2

In the cytosol, afterwards it is stored in vesicles

Question 3

For this reaction, name the substrates and products

What’s the enzyme?

Where is the enzyme (cell type and intracellular compartment)?

Answer 3

Substrates: choline + acetyl-CoA

Products: Acetylcholine + CoA-SH

Enzyme: choline acetyltransferase (ChAT)

Location: cytosol of cholinergic axon terminal

Question 4

What triggers acetylcholine release from the axon terminal?

Answer 4

Calcium signal

Action potential -> depolarized axon terminal -> calcium entry via voltage-gated calcium channels -> exocytosis of vesicles

Question 5

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?

Answer 5

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.

Question 6

Where does most of the acetyl-CoA used in formation of acetylcholine come from?

Answer 6

Glucose metabolism, only in long-term starvation will it come from other sources

Question 7

What is most capable of limiting acetylcholine synthesis?

What is NOT rate-limiting in ACh synthesis?

Answer 7

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.

Question 8

What inhibits choline uptake in cholinergic neurons?

Answer 8

Hemicholinium

Question 9

What are 6 steps in the cycle of vesicles going through uptake of their neurotransmitter, release, and back again?

Answer 9

1. Uptake of NT into vesicle
2. Formation of cluster of vesicles - reserve pool
3. Docking of vesicles into the active zone: there will not be actual physical contact between the vesicle and plasma membrane
4. Priming- vesicles become ready for calcium-induced fusion
5. Calcium signal -> fusion to lipid membrane
6. Vesicle is recycled

Sometimes they can touch the membrane without NT release in either the “kiss and stay” or “kiss and run” way

Question 10

Where are SNAPs? Where are VAMPs?

What do these acronyms stand for?

Answer 10

SNAPs are in the plasma membrane, VAMPs are in the vesicle membrane

SNAP = synaptosomal-associated protein

VAMP = vesicle-associated membrane protein

Question 11

What happens between VAMPs and SNAP-25 to prepare a vesicle for calcium stimulus (“priming”)?

Answer 11

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”

Question 12

What is synaptotagmin?

Answer 12

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

Question 13

What toxin is specific for the SNARE motif proteins used for the cholinergic neurons?

How does it work?

Answer 13

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

Question 14

What is the main difference between nicotinic and muscarinic acetycholine receptors?

What are their major inhibitors?

Answer 14

Nicotinic receptors are ligand-gated receptors

Muscarinic receptors are G-protein coupled receptors (GPCR’s)

Nicotinic is inhibited by curare, muscarinic by atropine

Question 15

Which type of GPCR is found in the M1 muscarinic acetycholine receptor? What are those GPCR effects (briefly, what’s the cascade)?

Answer 15

M1 = Gq

-> phospholipase C -> PIP2 -> IP3 -> Calcium signal

Question 16

Which type of GPCR is the M2 receptor?

Where in the body is it found, importantly?

What are its effects?

Answer 16

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

Question 17

What type of GPCR are M3 receptors?

Where are they found, importantly?

What is their function?

Answer 17

M3 = Gq

Found in the gastrointestinal tract

Participate in smooth muscle contraction and stimulation of gastrointestinal secretions

Question 18

How many subunits make up the nicotinic acetylcholine receptor?

Answer 18

5 subunits total (pentamer)

2 identical alpha subunits

then there is 1 each of beta, gamma, delta

Question 19

What is the direct effect of nicotinic acetylcholine receptors?

Answer 19

Sodium and potassium become permeable, resulting in membrane depolarization

In muscle, this leads to calcium being released from the sarcoplasmic reticulum

Question 20

What toxin is specific for only the muscular nicotinic acetylcholine receptors?

Answer 20

alpha-bungarotoxin (a snake venom)

Leads to paralysis

Question 21

What is an autoimmune attack of nicotinic receptors called?

What occurs?

What is the first symptom of this?

Answer 21

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

Question 22

How specifically is calcium released from the sarcoplasmic reticulum after nicotinic acetylcholine receptors are activated in skeletal muscle?

Answer 22

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

Question 23

Where in the body is the ryanodine (RYR) type 1 receptor?

Where is the RYR2?

Answer 23

RYR1: skeletal muscle

RYR2: heart muscle

Question 24

What condition results from mutation of the RYR1 receptor?

Answer 24

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)

Question 25

Which neurotransmitters are inactivated enzymatically?

Answer 25

Only acetylcholine is

Question 26

What is the significance of “pseudocholinesterases” - cholinesterases that are in the blood plasma, and not in the cholinergic nerve terminal?

Answer 26

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

Question 27

What is used to test for myasthenia gravis?

What about treatment?

Why?

Answer 27

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

Question 28

Why might cholinesterase inhibitors be used to treat glaucoma?

Answer 28

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

Question 29

What is the major family of irreversible cholinesterase inhibitors?

What are some examples?

How do they work?

Answer 29

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

Question 30

What is the normal plasma choline concentration?

What is “low affinity” choline uptake’s Km and location?

What about “high affinity”?

Answer 30

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)