Neurotransmission

B.Sc 2002

Archetypal neurotransmitter is acetylcholine;

We know more about cholinergic transmission than any other. However, cholinergic transmission is the exception not the rule in the CNS

+CH3 C OCH2CH2 N

CH3

CH3

CH3

O

acetylcholine

Acetylcholine can be very rapidly hydrolysed, that is broken down by reaction with water. It may be this characteristic that makes it suitable as a transmitter

+ +

+

CH3 C OCH2CH2 N

CH3

CH3

CH3

O

+CH3 C OCH2CH2 N

CH3

CH3

CH

O

HOH

choline

acetylcholine

acetic acid

H O2

Why is ACh a transmitter? What makes it suitable?

Ach is a quaternary amine; it has a nitrogen atom that is positively charged at one end of the molecule

Most compounds that are agonists at cholinergic synapses also have a positively charged nitrogen

+CH3 C OCH2CH2 N

CH3

CH3

CH3

O

acetylcholine

Some General Cholinergic agonists

Two types of cholinergic agonists;Nicotinic and Muscarinic

Examples of Specific Nicotinic agonists: Nicotine, Lobeline

N

N

N

CH3

Nicotine

Lobeline

O O

N

N

N

CH3N

N

CH3

Nicotine

Lobeline

O O

H

+

H

+

Free base at pH 7

Muscarinic agonists do not need positively charged nitrogen;

Nicotinic: ion channel openingMuscarinic: G-protein coupled

• M1; ‘neural’; slow epsps in ganglia; increase IP3,DAG

• M2; ‘cardiac’ decrease heart rate; decrease cAMP

• M3;’glandular’ increase secretion increase IP3

Atropine & scopolamine are antagonists at all muscarinic synapses

Transmitter release

Acetylcholine is found in synaptic vesicles (50 nm diameter), clustered around release zones in the presynaptic membrane.

Voltage-gated N- or P- type calcium channels are found in large numbers in the presynaptic membrane

Blockade of these channels (black widow spider venom, conotoxin) prevents transmitter release

After stimulation of axon, muscles normally show an ‘end plate potential’ that triggers AP. In quiescent muscles, spontaneous ‘miniature’ end plate potentials are observed.

10-30 ms

-70 mv-65 mv

Time of arrival ofaction potential inafferent terminals

Motoneuronemembranepotential

EPSP

-90 mv

-40 mv

Time of arrival ofaction potential atneuromuscluar junction

Musclemembranepotential

EPP

Action potential threshold

-90 mv

-85 mv

Musclemembranepotential

spontaneous miniature end-plate potentials,always of fixed sizes

Fact that mepps were quantized (2 mV, 4mV, 6 mV, not e.g. 3.5 mV, was evidence that transmitter was released in packets, or quanta. This led to idea that transmitter was released from vesicles. Even in absence of stimulation, some vesicles were releasing transmitter

Methods to study transmitter release mechanisms

• Toxins (mostly proteases) eg botulinus toxin, tetanus toxin

• Capacitance measurements of secreting cells

• Intracellular ion-sensitive dyes

• Mutation of intracellular vesicular & membrane proteins

Synaptic docking proteins

On vesicle:

synaptotagmin (sytg), synaptobrevin (syb)

On plasma membrane:

SNAP-25, syntaxin (sytx)

In cytoplasm:

NSF (contains -SNAP)

Synaptic Vesicle

SYB

SYTX

SYTG

SNAP-25

Plasma membrane

NSF

Ca channel++

Synaptic Vesicle

SYB

SYTX

SYTG

SNAP-25

Plasma membrane

NSF

Ca channel++

Docking complex formedVesicle separate

Syb: synaptobrevin, Sytg: synaptotagmin,sytx: syntaxin

Synaptic Vesicle

SYB

SYTX

SYTG

SNAP-25

Plasma membrane

NSF

Ca channel++

Docking complex formed Fusion complex formed

Synaptic Vesicle

SYB

SYTX

SYTG

SNAP-25

Plasma membrane

NSF

Ca channel++

Calcium enters; activates sytg

Synaptic Vesicle

SYB

SYTX

SYTG

SNAP-25

Plasma membrane

NSF

Ca++

sytg interacts with sytx;Transmitter release

SYB

SYTX

SYTG

SNAP-25

Plasma membrane

NSF

Ca++

Transmitter can ‘leak’ from fusion complexes before proper calcium triggering

Spontaneous release can occur from fluctuations in intracellular calcium

Clathrin

DockedExocytosis

Endocytosis

Migration

Refilling

Pre-fusion

Storage pool

Vesicle Life-Cycle

Studies on ACh receptors have used the electric eel‘torpedo’ as it contains very large amount of receptor protein in the electroplax organ.

Complete ACh channel

outside cell

inside cell

NH2

COOH

One ACh channel subunit

subunit

pore

subunit seen from above

five subunitsarrangedin a ring

long (210 residues) N-terminalhydrophilic extracellularregion pointing towardsACh release site

subunit

pore

subunit

Nicotinic ACh Receptor

ACh Binding site

2M 2M

2M4M

4M4M

4M

4M

2M

2M

Lipophilic bonding

Transmitter inactivation

• ACh uses enzymic breakdown as mechanism of inactivation

• All other transmitters use reuptake as inactivation mechanism

• UNSOLVED PROBLEMS:

• How does ACh unbind from binding site?

• What is actual mechanism of allosteric distortion?

Cholinegic antagonists

• Depolarisation blockers eg suxamethonium

• Do not unbind from receptor, leave muscle depolarised and unable to fire second AP

• Curare-like agents bind to receptor but do not open ion channel

N

NO

O

OH

OH

CH O3

H

H

H

3CH3CH

3CH

Tubocurare

+

+

Distance apart of two ACh N binding sites?+

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are needed to see this picture.