CHOLINERGIC TRANSMISSION

Maj Jackson Yamvwa

 Learning Objectives
1. To explain the synthesis and release of Acetylcholine

2. To discuss the electrical events in transmission at cholinergic synapses

3. To explain the effects of drugs on cholinergic transmission

 Expected Learning Outcomes

• At the end of this lesson, you should be able to:

1. Explain the synthesis and release of Acetylcholine
2. Discuss the electrical events in transmission at cholinergic
synapses
3. Describe the effects of drugs on cholinergic transmission
 Introduction
• Acetylcholine (ACh) is the transmitter at:
Neuromuscular Junction (NMJ)

Autonomic ganglia

Postganglionic parasympathetic nerve ending

Various synapses in the CNS

Biosynthesis of Acetylcholine
– Choline Acetyltransferase (ChAT)

– Choline and Choline Transport

 Choline Acetyltransferase
• This is the enzyme that catalyses the synthesis of ACh.
• It catalyses the acetylation of choline with acetyl CoA
• It is synthesized with the perikaryon and transported along the length
of the axon to its terminal.
• Acetyl CoA is synthesized in the mitochondria.
• Choline is taken up from the extracellular fluid into the axoplasm by
active transport.
• The final step in the synthesis occurs within the cytoplasm, following
which most of the ACh is sequestered within synaptic vesicles.
 Choline Acetyltransferase
• The availability of choline is critical to the synthesis of ACh.

• Choline is primarily derived from the diet or, secondarily,

from recycling of choline.
• There is little de novo synthesis of choline in cholinergic
neurons.
 RE-UPTAKE
• Once ACh is released from cholinergic neurons in response
to an action potential, ACh is hydrolysed by AChE to acetate
and choline.
• Much of the choline is taken up at cholinergic nerve
terminals and reused for ACh synthesis.
• Under many circumstances, this reuptake and availability of
choline appear to be rate limiting in ACh synthesis.
 RE-UPTAKE
• Once ACh is released from cholinergic neurons in response
to an action potential, ACh is hydrolysed by AChE to acetate
and choline.
• Much of the choline is taken up at cholinergic nerve
terminals and reused for ACh synthesis.
• Under many circumstances, this reuptake and availability of
choline appear to be rate limiting in ACh synthesis.
 Choline Transport and Transporters
• There are three mammalian transport systems for choline.
• All three are transmembrane proteins with multiple TM
segments.
1. CHT1 (SLC5A7)
– This is a high affinity, Na+/Cl- dependent choline
transporter.
– It is present on presynaptic membranes of cholinergic
neurons.
– This system provides choline for ACh synthesis
 Choline Transport and Transporters
• CTL1 (SLC44A)
• This is a low affinity, Na+-independent transporter
• It is widely distributed and appears to supply choline for
phospholipid synthesis (e.g., phosphatidyl choline,
sphigomyelin).
 Choline Transport and Transporters
OCT2 (SLC22A2)
• This is a lower-affinity, Na+-independent transporter.
• It is a nonspecific organic cation secretory transporter found
in the renal tubules, hepatocytes, the choroid plexus, the
luminal membrane of the brain endothelium, and synaptic
vesicles from cholinergic neurons.
• Its roles remains unclarified.
 Choline Transport and Transporters
• All these transporters are inhibited by

hemicholinium but at distinct concentrations in the

same order as their affinities for choline.

 Summary of Synthesis of Acetylcholine
• ACh is synthesized in the cytoplasm from acetyl-CoA and choline
through the catalytic action of the enzyme choline acetyltransferase
(ChAT).
• Acetyl-CoA is synthesised in mitochondria, which present in large
amounts in the nerve ending.
• Choline is transported from the extracellular fluid into the neuron
terminal by sodium-dependent membrane choline transporter (CHT1
[SLC5A7])
• Hemicholinium-3 (HC3), also known as hemicholine, is a
drug which blocks the reuptake of choline by the high-affinity
choline transporter at the presynapse.
• The reuptake of choline is the rate-limiting step in the
synthesis of acetylcholine;
• Hence, hemicholinium-3 decreases the synthesis of
acetylcholine.
• It is therefore classified as an indirect
acetylcholine antagonist
• Acetylcholine is synthesized from choline and a donated acetyl group
from acetyl-CoA, by the action of choline acetyltransferase (ChAT).
• Thus, decreasing the amount of choline available to a neuron will
decrease the amount of acetylcholine produced.
• Neurons affected by hemicholinium-3 must rely on the transport of
choline from the soma (cell body), rather than relying on reuptake of
choline from the synaptic cleft.
• Hemicholinium-3 is highly toxic because it interferes with

cholinergic neurotransmission.

• The LD50 of hemicholinium-3 for mice is about 35 μg.

 Synthesis of Acetylcholine
Choline Acetyltransferase
• Acetyl-CoA + Choline Acetylcholine +
CoA

• The choline transporter can be blocked by a group of

research drugs known as hemicholinium.
• Different transporters are inhibited at varying
concentrations by hemicholinium.
Synthesis and Storage of ACh
 Storage of ACh
• ACh is transported into synaptic vesicles by the VAChT.

• The transporter uses the potential energy of a proton

electrochemical gradient that a vacuolar ATPase establishes.
• The transport of protons out of the vesicle is coupled to
uptake of ACh into the vesicle against a concentration
gradient.
 ACh Storage
• The process is inhibited by the noncompetitive and
reversible inhibitor vesamicol, which does not affect the
vesicular ATPase.
 Vesicles and SNARE Proteins
• Vesicles are concentrated on the inner surface of the nerve
terminal.
• Each vesicle has membrane bound proteins known as
Vesicle - associated Membrane Proteins (VAMPs) called v-
SNAREs, especially synaptobrevin.
• On the inside of the terminal neuron membrane, there are
membrane bound proteins known as synaptosome-
associated proteins (SNAPs) called t-SNAREs.
• The commonly known t-SNAREs are syntaxin and SNAP-25.
 Summary of ACh Storage

• Once synthesised, Ach is transported from the cytoplasm

into the storage vesicles by a vesicle-associated

transporter (VAT) that is driven by proton efflux (VAChT).

• This antiporter can be blocked by the research drug known

as vesamicol.
 Release of ACh
• The release of Ach from the vesicles is dependent on
extracellular calcium.
• It occurs when an action potential reaches the terminal and
triggers sufficient influx of calcium ions.
• Ca2+ interacts with the VAMP synaptotagmin on the vesicle
membrane and triggers:
 Fusion of the vesicle membrane with the terminal membrane
 Opening of a pore into the synapse.
• Opening of the pore results in exocytotic expulsion
 Release of ACh

• The ACh vesicle release process is blocked by botulinum

toxin through the enzymatic removal of two amino acids

from one or more fusion proteins.

 Effect of Botulinum and Tetanus Toxins on
Release of Acetylcholine
• The release of Ach and other NT by exocytosis through the
prejunctional membrane is inhibited by botulinum and
tetanus toxins from Clostridium.
• Clostridium toxins are transported into the cytosol through
endocytosis.
• They consist of disulphide-like heavy and light chains.
• The light chain is a Zn2+ -dependent protease that becomes
activated in the cytosol.
Effect of Botulinum and Tetanus Toxins on Release of
Acetylcholine

• Upon activation, it hydrolyses components of the core or SNARE

complex involved in exocytosis.

• The various serotypes of botulinum toxin digest selected proteins

in the plasma membrane (Syntaxin and SNAP-25) and the

synaptic vesicle (synaptobrevin).

 Effect of Botulinum and Tetanus Toxins on
Release of Acetylcholine
• Tetanus toxin is transported in retrograde fashion up the motor
neuron to its soma in the spinal cord.
• From there, the toxin migrates to inhibit neurons that synapse with
the motor neurons and blocks exocytosis in the inhibitory neuron.
• The block of the release of the inhibitory neurotransmitter gives rise
to tetanus or spastic paralysis.
 The Fate of Ach at Cholinergic Synapses
• After release from the presynaptic terminal, Ach molecules bind
to and activate an acetylcholine receptor (Cholinoceptors).
• Eventually, all the Ach released diffuses with range of an
Acetylcholinesterase (AChE).
• AChE splits (hydrolysis) into choline and Acetate.

• Neither of these has significant transmitter effect.

 Cholinesterases
• For ACh to serve as a neurotransmitter, it must be removed or

inactivated within the time limits imposed by the response

characteristics of the synapse.

• This effect is achieved by the enzymes known as

Cholinesterases.
 Cholinesterases
• There are two forms of cholinesterases.
1. Acetylcholinesterase (AChE)

2. Butyrylcholinesterase (BuChE)
• Both are serine hydrolases which hydrolyse
ACh and other esters.
• They have different locations and specificities.
 Differences between AChE and BuChE

Enzyme Location Substrate Specificity

AChE Basement membrane at ACh and closely related
cholinergic synapses compounds

Erythrocytes

BuChE Solution form in plasma Less specific:

butylcholine, other esters
Liver (e.g., succinylcholine
Elsewhere and procaine)
AChE Binding Sites
Ach binding to AChE

Sites of Action of Anticholinesterase

Drugs
They may bind to either one or both sites with
different durations of actions
 Cholinesterases: Enzyme Action
• Hydrolysis of ACh occurs in three steps:
1. ACh binds to the enzyme
2. The acetyl group is transferred to a serine OH in the esteratic site
of the enzyme, resulting in a transiently acetylated enzyme plus
free choline
3. Hydrolytic cleavage of the serine-acetyl bond releases acetyl
group.
• At fast synapses, such as those at NMJ and in ganglia, but
not at slow one (cardiac muscle, smooth muscle, glands),
AChE hydrolyses the released ACh in about 1ms.
 Anticholinesterases
• There are three main groups of anticholinesterases.
• They are classified on the basis of their duration of action depending
on how they interact with AChE
• Short acting: simple reversible association with the anionic site of the
enzyme (e.g. edrophonium)
• Medium acting; interact with the serine hydroxyl at the active site give
a carbamylated product, which is only slowly hydrolysed (e.g.
neostigmine, physostigmine, pyridostigmine)
• Irreversible acting; these irreversibly phosphorylate the serine
hydroxyl group. Most are organophosphate compounds with
generalised toxic effects
Anticholinesterases