Lecture 4 - Acetylcholine Neurochemistry + Muscarinic and Nicotinic Receptors

sites of neurotransmission

• All ganglia of the ANS

• Postganglionic parasympathetic junctions

• Neuroskeletal muscular junction

• Adrenal medulla

• Postganglionic sympathetic nerve junctions at sweat glands

• CNS

structure of acetylcholine

consists of ester (blue), ethylene bridge (green), and 4° ammonium (yellow)

chemical properties of ACh (acid/base, potential binding interactions, stability)

acid/base: neutral

binding interactions: H-bond acceptor (ester), ion-dipole and ionic (4° ammonium)

stability: unstable (due to ester)

neutransmitter function steps

1. biosynthesis in presynaptic neuron

2. storage in presynaptic neuron

3. release to synapse

4. receptor activation (pre- or post- synaptic) (generation of second messenger after binding to receptor)

5. termination of neurotransmission in synapse (metabolic breakdown/reuptake)

substrate

molecule that undergoes a chemical change or reaction, often being acted upon by an enzyme or catalyst

enzyme

biological catalyst, typically a protein, that speeds up specific biochemical reactions in living organisms without being consumed

biosynthesis of ACh chemistry steps

co-factor

non-protein substance (like a metal ion or organic molecule) essential for an enzyme's catalytic activity, helping it bind substrates or facilitate reactions by stabilizing structures or transferring groups

why do we need to store ACh

1. Maintain a ready supply of the neurotransmitter (acetylcholine)

2. Protect neurotransmitter (acetylcholine) from premature enzymatic degradation

second messenger of action potential

calcium (and its influx)

what makes nicotine and muscarine like ACh

• available group to be ionized

• group that can do H bonding

Nicotinic Acetylcholine Receptors (nAChRs)

ligand-gated ion channels activated by the neurotransmitter acetylcholine, famously also by nicotine, playing crucial roles in the central and peripheral nervous systems

ligand

molecule, ion, or atom that binds to another, often larger, molecule (like a protein or central metal atom) to form a complex, acting as a signaling molecule in biology

ligand-gated ion channel

transmembrane protein that forms a pore, opening or closing (gating) in response to a specific chemical signal (ligand), like a neurotransmitter or second messenger, binding to it, thereby controlling the flow of ions across a cell membrane

how does ACh work on nicotinic receptors

1. Originally, The channel pore is closed, preventing ion flow

2. 2 ACh molecules bind to the two binding sites on the receptor

3. binding acts as a "1st messenger" in which receptor undergoes a conformational change and opens

4. Ions flow through: primarily Na⁺ (sodium) flows INTO the cell and K⁺ (potassium) flows out; Ca²⁺ (calcium) may also enter, acting as "2nd messengers"

5. ion influx depolarizes the cell membrane, triggering cellular responses

6. when ACh disassociates, channel closes

desensitized nAChR state

Ligand is still bound to the receptor but the channel has closed despite ACh still being attached; he receptor becomes temporarily unresponsive and prevents overstimulation

where are nAChR located

all autonomic ganglia, neuromuscular endplate of striated muscle, adrenal medulla

LGIC stands for…

ligand-gated ion channel

where does ACh binding on nAChR actually occur

on the two alpha-subunits

Nm (Nicotinic, neuromuscular) receptors

specialized ligand-gated ion channels found at the neuromuscular junction (NMJ) of skeletal muscles, activated by acetylcholine (ACh) to trigger rapid muscle contraction via end-plate depolarization

Nn (Nicotinic, neuronal) receptors

ligand-gated ion channels for fast neurotransmission in the autonomic nervous system (ANS) and central nervous system (CNS), mediating responses to acetylcholine (ACh) and nicotine from neuron to neuron

general structure of GPCR

• Seven transmembrane helices

• Extracellular binding domain (neurotransmitter binds here)

• Intracellular effector domain (interacts with G-protein and activates it)

• Heterotrimeric G-protein (dissociates upon activation)

• Effector (activated by the G-protein)

• Second messenger(s) - produced by activated effector

• Biological response

how does ACh work on muscarinic (GPCR) receptors

1. Binding of ACh to the m1, m3, and m5 receptors results in the activation of G-proteins (Gq and G11)

2. G protein’s alpha site activate the effector phospholipase C (an enzyme)

3. Phospholipase C catalyzes the hydrolysis of phosphoinositide to form the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG)

4. second messengers cause an increase in calcium influx (into the cell) at the endoplasmic reticulum, as well as protein kinase C activation

5. cell response occurs

1st messenger in GPCR signaling for ACh

ACh! it also acts as the ligand

what happens when ACh binds to M2 and M4 receptors?

1. Activation of the Gi G-protein inhibits the effector adenylate cyclase (AC, an enzyme) that causes a decrease in the conversion of ATP to cAMP, the second messenger that normally produces a biological response (generally muscle contraction);There is therefore less cAMP (second messenger) available and there is a corresponding decrease in muscle contraction.

2. Activation of the Gi or Go G-protein that regulates K+ channels (typically opens it)

effector

substance or molecule that binds to a target (like an enzyme or receptor) to change its activity, thereby producing a cellular or physiological response

what interaction(s) occur between ACh and muscarinic transmembrane unit 1

ion-dipole (as ion) —> presence of threonine with secondary alcohol + near ionized amine

what interaction(s) occur between ACh and muscarinic transmembrane unit 2

ion-dipole (as ion) —> presence of serine with primary alcohol + near ionized amine

what interaction(s) occur between ACh and muscarinic transmembrane unit 3

ion-dipole (as ion) —> presence of tyrosine with phenol + near ionized amine
ionic —> presence of asparagine with ionized amide + near ionized amine

what interaction(s) occur between ACh and muscarinic transmembrane unit 4

n/a

what interaction(s) occur between ACh and muscarinic transmembrane unit 5

H-bonding —> presence of threonine with secondary alcohol + near ester

what interaction(s) occur between ACh and muscarinic transmembrane unit 6

H-bonding —> presence of tyrosine with phenol + near ester
ion-dipole (as ion) —> presence of tyrosine with phenol + near ionized amine

what interaction(s) occur between ACh and muscarinic transmembrane unit 7

ion-dipole (as ion) —> presence of tyrosine with phenol + near ionized amine

Mechanism of AChE hydrolysis of acetylcholine

1. Acetylcholine enters the active site of AChE

2. The ester bond of ACh (connecting acetyl group to choline) is positioned near SER 200

3. SER 200's oxygen attacks the carbonyl carbon of the acetyl group, breaking the bond between acetyl and choline

4. Choline (HO-CH₂-CH₂-N(CH₃)₃) leaves as the first product, while Acetyl group stays attached to SER 200

5. Water attacks the acetyl group attached to SER 200, which breaks the bond between acetyl and SER 200

6. Acetic acid (CH₃COO⁻) leaves as the second product

7. Enzyme is free and ready to work again