1 - PHA 4107 Week 1 Molecular Pharm GCPRs and Ionotropic receptors (Jan 11)

Examples of GCPRs (G-coupled proteins)

• Acetylcholine (muscarinic)

• Adrenaline (adrenergic)

• 5-hydroxytryptamine (serotonin)

• opiate receptors

• dopamine receptors.

• Many peptides (peptide hormone receptors)

• smell (olfactory receptors)

• sight (rhodopsin-photon transduced receptor)

What was the first receptor (GCPR) to be characterized?

Beta-adrenergic receptor

What are GCPRs (G-coupled proteins)?

• They bind to GTP

• Hormones work through ____’s

• ¼ of all prescription drugs work through ___

What is the basic structure of a GCPR?

• 400-500 AA in length

• 7 transmembrane helical segments (serpentine receptors) —> highly conserved structure

• C3 is the most important helices because it plays a role in G-protein binding

What does a point mutation in the C3 loop do to GCPR signalling?

It destroys signalling because G-protein can’t bind

How do GCPRs work?

Summary:

• ligand binds to receptor and changes shape to bind to G protein

• G-protein activated —> exchanges GDP for GTP

• Ligand dissociates receptor associates with effector to make a second message

What is an RGS? What does it do?

• regulator of G-protein signalling

• Activates GTPase activity to deactivate subunit

What is receptor desensitization?

Ligand response decreases with time —> can be fixed by removing ligand and regaining response

How are GPCRs desensitized ? Why?

It prevents overstimulation of the receptor

• GRK phosphorylation receptor

• Arestin protein binds to receptor to prevent G-protein binding

• Desensitization is achieved

G-protein summary

• intermediates between receptor and effector molecules

• Bind to GDP and GTP

• 3 subunits: a, b, y

• a subunit awards specificity

• b and y subunits associated in heterodimeric complex

How can a specific receptor only control one kind of effector

Not completely understood but not all G-proteins are identical. There are several different a subunits. Inhibitory and stimulatory GCPRs have same target effector (ex. GS and Gi stimulate and inhibit adenylate cyclase).

• allows different receptors to exert opposite effects on a target effector enzyme in the presence of agonists

How do G-proteins activate different effectors?

1. Gas: activates adenylate cyclase

2. Gaq: activates phospholipids C

3. Gai: inactivates adenylate cyclase but activates cyclic GMP phosphodiester-ase

What are ionotropic receptors / ligand gated ion channels?

• channel-linked receptors: fast post-synaptic responses

• Channels are activated by directly bound ligand

  • Ex. Neurotransmitters

    • GABA receptor

    • Nicotinic Acetylcholine receptor

    • 5-HT3 (serotonin) receptor

What is the structure of a ligand gated ion channel

• 4 main subunits (a,b,y,s)

• Each subunit spans the membrane 4x (20 membrane spans total)

• M2 subunit always faces the core

The excitable membrane

• inner membrane is -70mV

• Influx of +ve ions at nerve synapse will result in DEPOLARIZATION and the establishment of an ACTION POTENTIAL

• Efflux of +ve ions or influx of -ve ions repolarizes membrane

Where can acetylcholine and ionotropic acetylcholine receptors found?

• neurotransmitter found at the post-synaptic membrane of neurons

• Receptor found in muscle at the neuromuscular junction (action potential here activates the opening of Ca2+ channels resulting in muscle contraction)

How does cell signalling work?

The ionotropic receptors (ion channels) depolarize the membrane when synapse occurs. Voltage gates Na+ channels propagate the current / signal

• ex. Na+ and K+ increase permeability induced by acetylcholine (muscle) and glutamate (CNS)

  • Result: depolarization and formation of action potential

  • Acetylcholine binds to straighten the a-helices in the channel, allowing 10^7 ions pe second through the pore

How do you have ion selectivity?

Point mutations of the critical AA in the M2 helix of an ion channel changes specificity from cation to anion selectivity

How many subunits does a nicotinic receptor subunit have?

5 subunits per receptor —> homomeric

How many subunits does a glutamate receptor have?

4 subunits per receptor

NMDA receptors (N-methyl-D-aspartate)

• located at post-synaptic density region of excitatory synapses

• Permeable to K+ and Ca2+ ions

• NR1 and NR2 GLU subunits are associated in a tetrameric orientation in the membrane

AMPA/glutamate receptor

• heteromeric combinations of GLU NR1-4 subunits

• Mediates fast excitatory synaptic transmission

• Increased Na+/K+ cation permeability (NOT Ca2+)

GABA receptors

• ligand gated chlorine channels (ONLY CHLORINE!!!)

• At post-synaptic membrane of GABA-ergic synapses

• INHIBITORY NEUROTRANSMITTER!!! (Other ex. Barbiturates and benzodiazepines)

Nicotinic / ACh (acetylcholine) receptor

• excitatory increase in Na+ and K+

• Present in post synaptic neuron and at NEUROMUSCULAR JUNCTION

• Clustering is mediate via rapsyn and utrophin

PSD proteins

NR2 C-terminus mediates interaction with a large multiprotein complex in which the main protein is PSD-95

High efficacy agonists vs low efficacy agonists

• most receptors have same conductance (flow rate) for different agonists

• Agonists differ in the channel lifetime that they induce

Ligands (agonists (b) and antagonists (a))

1. High efficacy: large amount of receptors are active at any given time (b>a)

2. Low efficacy: fewer receptors are active at a time (a>b)

3. Antagonist: b=0