1

Neuroscience with Pharmacology 2

Neuromuscular Junction 2: Pharmacology

1. Principles and methods for studying ACh pharmacology at the NMJ

2. Targets for drug action at the NMJ (synthesis, storage, release, action, inactivation)

3. Drugs affecting postsynaptic (nicotinic) ACh Receptors

Neuromuscular Junction - Pharmacology

1. Principles and methods

Acetylcholine

“Quaternary” nitrogen

+

Targets in neuromuscular pharmacology :

Synthesis

Storage

Release

Action

Inactivation

Drugs acting at the NMJ are useful as:

Experimental tools

Clinical treatments

2

• Target specificity/Ligand specificity

• Agonist/Antagonist

• Dose/Response

• Efficacy (EC50/IC50)/Affinity (KD)

• Competitive/Non-competitive

• Clinical/Non-clinical uses

• Side-effects

When “doing drugs” a good pharmacologist needs to know:

Biological response (->efficacy, EC50)

Ligand binding (->affinity, KD)

Drug assays at the NMJ are based on:

Several techniques can be used to assaythe effects of drugs at NMJ’s

1. Muscle contraction

2. Intracellular EPP recording

3. Iontophoresis/patch clamp

4. Ligand binding

Measuring muscle contractions in response torelease of neurotransmitter by nervestimulation….

Atropine d-Tubocurarine

Action potential

…add µ-conotoxinX

…add d-tubocurarine

Measuring EPP’s….

3

Testing drugs on ACh receptors

Iontophoresis

Bath application

Patch clamp

I

V

Ch.2

2 mV

30.00 ms

s

10 µM ACh

2 mV

60 s EC50

Ligand binding visualised…

α-bungarotoxin

Control

10µm α-BTX

But normally measured chemically (eg radioactively-labelled ligand)…

Drugs that block ACh Receptors (antagonists)

Example:d-tubocurarine

Example:α-bungarotoxin

Reversible

Irreversible

2. Targets for drug action at the NMJ

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α-bungarotoxind-tubocurarineatracuriumsuxamethonium

edrophoniumneostigminesarin

hemicholinium

vesamicol

botulinium (A-D)

Targets of drug action at the neuromuscular junctionDrugs that inhibit transmitter release

Botulinum toxins = ‘SNARE’ blockers P/Q Ca2+ channel blockers

Ca2+x

x

ω-agatoxin/ω-conotoxin

α-latrotoxin

BEFORE

K channel blockers and some toxins enhance transmitter release

AFTER

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

-1.0

-1.5

mV

AC

1

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200s

During

0 Ca +Ca +4AP TTXDirectDirect +Mg +4AP

3. Drugs affecting postsynaptic (nicotinic) ACh Receptors

ACh + R ACh-R ACh-R*

ACh-R0

General scheme for Agonist-Receptor Interaction (illustrated by ACh binding to its Receptors)

“desensitized”

bound activated

[ACh].[R]

[ACh-R]KD = = ~ 80 µM

5

8 nm

The nicotinic ACh Receptor at NMJ

/ε

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/LIPIDS/transport.html

nACh Receptor Pharmacology - Neuromuscular Junction

Agonist Antagonist/blocker

nicotine d-tubocurarine (curare)

acetylcholine α-bungarotoxin

carbachol atracurium

decamethonium pancuronium

suxamethonium (I) suxamethonium(II)

6

http://en.wikipedia.org/wiki/Quaternary_ammonium_muscle_relaxants

ACh

Decamethonium Pancuronium

ACh

ACh

Na+

K+

dTC

ACh

dTC

dTC

ACh ACh

ACh ACh

ACh dTC

ACh

Na+

K+

ACh

7

ACh

Na+

K+

αBTX

ACh

αBTX

αBTX

ACh ACh

ACh AChACh

ACh

ACh

AChACh

Not broken down by AChE (nmj) Broken down by BuChE (blood plasma) Short lasting Not counteracted by cholinesterase inhibitor Used clinically for brief muscle relaxation “Depolarising blocker”

2

2

AcetylcholineSuxamethonium

dTC dTC neo sux sux sux neo direct

Sux

Sux

Na+

K+

Sux

Na+ Na+

K+

+ +

8

Sux

Na+

+

Na+

K+

+

Sux

+

Na+ Na+

K+

SuxNa+

K+

Sux

Summary

1. Every step in the cycle of ACh synthesis, storage, release, activation and inactivation is a potential target for drug action at the NMJ.

2. Drugs affecting release 4-AP, α-latrotoxin, botulinum toxin, agatoxin

3. Drugs that block ACh Esterase:• edrophonium, prostigmine, neostigmine, sarin• TEA, 4-AP, α-latrotoxin, botulinum toxin, agatoxin

4. Drugs affecting storage and synthesis :• hemicholinium, vesamicol• AF64A

5. Drugs affecting ACh Receptors:• carbachol, decamethonium, suxamethonium• tubocurarine, α-bungarotoxin, atracurium

6. Clinical uses of drugs acting at the NMJ:• muscle relaxants as adjunct to anaesthesia in surgery• treatment of neuromuscular disease