What are the 6 steps involved in cell-cell communication?
Synthesis of signalling molecule
Release of signalling molecule
Transport to site of Target Cell
Detection of signal by specific receptor
Alteration in cell function
Removal of signal & termination
What are the 3 types of cell-cell communication?
Define Autocrine communication and give an example
Autrocrine - Cell responds to its own released signal, so signal controls activity of Signalling Cell.
e.g Epinephrine/Presynaptic a2-adrenoceptors - Inhibits neurotransmitter release.
[Via Plasma Membrane-Attached Proteins:]
- Or, the signal's not released but is tethered to the plasma membrane of the signalling cell.
e.g Ephrins/Ephrin Receptors
Define Paracrine communication and give an example
PARACRINE - Soluble signal links cells in close proximity
e.g ACh (acetylcholine) release from neurone at neuromuscular junction
Define Endocrine communication and give an example
ENDOCRINE - Signals (hormones) act on target cells distant from their site of synthesis
e.g Insulin secretion by ß-cells in pancreas
What 2 types of receptor systems receive extracellular signals?
- Intracellular Receptors
- Plasma Membrane Receptors
Give an example of an Intracellular Receptor and give an account of its' mechanism of action.
e.g Steroid & Thyroid hormone receptors
- Hydrophobic signal molecule bound to carrier in blood
- Crosses plasma membrane at site of action
- Binds receptor protein in cytosol
- Active receptor translocates to nucleus
- Active receptor binds DNA, acts as transcrition factor
Give an example of a Plasma Membrane Receptor and give an account of how it works.
e.g Receptors for small, charged molecules (epinephrine/adrenaline, histamine)
- Hydrophilic ligand can't cross membrane
- Binds to plasma membrane receptor protein
- Active receptor generates intracellular chemical signal (second messenger)
What determines how cells respond?
How do receptors function?
- Specifically detecting/binding to initial signal (AGONIST)
- Coupling agonist-bound receptor to the correct intracellular signalling pathway
- Amplifying agonist binding event into a detectable intracellular response
Signal —► Reception —►Transduction —►Response
[Between R and T is Amplification]
Give an example of a fast onset signal transduction.
Skeletal muscle (millisecs)
Give an example of a slow onset signal transduction
Heart muscle (several secs)
Why do signal transduction pathways need to be terminated?
- They would last forever
- Each stage requires a mechanism to terminate it
How does skeletal muscle respond to the agonist ACh and what receptor binds ACh?
- Depolarisation (on graph, steep rise, then gradual fall to resting potential)
- Rapid in onset (Millisecs)
- Nicotinic ACh receptor
How does cardiac muscle respond to the agonist ACh and what receptor binds ACh?
- Hyperpolarisation (On graph, steep fall, then rises slowly to R.P)
- Slow in onset (secs)
- Muscarinic ACh receptor
What are the 3 general principles of receptor structure?
1. Extracellular - Ligand binding
2. Transmembrane - Mostly hydrophobic
- Key charged residues control binding/ion passage
- a-helical structure
3. Intracellular - Interaction with signalling pathways
- Site of feedback control
What are the 4 classifications of plasma membrane receptors?
1. Ligand-gated ion channels
- e.g nicotinic ACh
2. Receptors that have enzymatic activity
- e.g Receptor Tyrosine Kinases (Insulin, platelet-derived growth factor)
3. Receptors non-covalently associated with enzymes
- e.g gp130 associates with Janus Kinases (JAKs)
4. G-Protein Coupled Receptors
- e.g Adrenoceptors
Are ligand-gated ion channels fast or slow in onset, and why?
LGIC function at fast synapses (millisec).
Rapid onset & rapid termination necessary for proper function.
Name a prototypical LGIC receptor
Nicotinic Acetylcholine Receptor
Where is ACh a key neurotransmitter?
At the neuromuscular junction
What happens at the neuromuscular junction?
- Nerve Impulse
- Vesicle fusion with membrane
- Release of acetylcholine into cleft
- ACh in cleft goes from 10nM —►500µM in 1 millisec
Decribe the effect of ACh on the post-synaptic membrane
- ACh receptors transiently increase Na+ influx and K+ efflux
- Resulting depolarisation opens voltage-gated sodium channels
- Leads to action potential & muscle contraction
- Must be fast ON/OFF to allow complex movement
What type of channel is nicotinic ACh receptor?
A non-selective cation channel, which transports all cations to a similar degree
What is nAChR common in?
The electroplaxes (voltage-generating cells) within electric organs of electric fish & rays
What does nAChR bind with high affinity?
Some neurotoxins e.g:
What allowed for the purification of nAChR?
It's high abundance in electric organs & high affinity for toxins.
What was the first channel to be purified and cloned?
nAChR (nicotinic acetylcholine receptor)
What is the structure of the muscle nAChR?
Pentamer (5 subunits) - (a1)2ß1γδ subunits
Subunits cluster to form a ring
Creates a pore in membrane to allow ion passage
Within the subunits of the muscle nAChR, what confers stability?
Glycosylation & disulphide bonds
Where is the ACh binding site within the nAChR subunits?
What are the 3 stages involved in the molecular basis of a channel opening?
- Resting State
- ACh binds
- Channel transiently activated
How many TM-Spanning helices are there in nAChR subunits?
There are 4 TM-Spanning helices - M1-M4, and are all a-helices
Which TM-Spanning helice is orientated into the channel pore?
What does each subunit have in their M2 helix, and what does this do?
A conserved 'leucine', which lines the centre of the pore to form a "gate".
Which residues in a channel pore provide electron pairs, and what are these pairs?
Specific Glu and Thr residues.
They attract cations (Na+, K+)
They exclude anions (Cl-)
During the resting state of channel opening, what do leucines do to the pore?
Leucines block the pore, so the gate is closed.
During the second stage of channel opening, where does ACh bind?
2 ACh molecules bind to hydrophilic extracellular regions of the receptor.
During the 3rd stage of channel opening, what does ACh binding cause to happen?
- ACh binding causes helices to tilt away from each other for approx. 1 millisec.
- Leucines no longer block pore so gate opens
- Cations pass down gradient into cell
- Helices move towards each other again - gate closes, ACh dissociates
How many a and ß genes exist and what does this mean?
9 a genes
4 ß genes
Several different channel combinations exist.
Cell type-specific expression.
e.g Muscle = (a1)2ß1γδ
Major brain subtype = (a4)2(ß2)3
Properties of each channel are distinct
Give examples of the types of anaesthetics that nAChR can bind and what effect does this binding have?
Isofluorane, halothane, nitrous oxide.
Reduces channel opening time
Give examples of what other LGIC nAChR subunits are related to.
AMPA, GABAA, 5HT3 receptors