Receptor Theory Flashcards
What are the four classifications of receptors?
1) Ligand gated ion channel
2) G protein coupled receptors
3) Kinase linked receptors
4) Nuclear receptors
What model can be used to explain efficacy?
The ‘lock and key’ model - serveral keys may fit but not all of them will work and cause an effect
What is meant by affinity?
The strength of interaction - dependent on the forward rate of association as well as the backward rate of dissociation
High affinity means drugs associate strongly
How is occupancy calculated?
No of receptors occupied / total number of receptors
Can you rely on measuring response as a way of measuring occupancy?
No - although response may increase with occupancy the two are independent of each other.
Occupancy is related to affinity
Radio ligand binding assays can be used to measure what?
The affinity of a drug
Describe how to perform a radio ligand binding assay
1) Prepare cells eg use detergent and centrifugation
2) Aliquot membrane out onto filters
3) Add the radiolabel at different concentrations and equilibrate
4) When equilibrated remove the unbound drug by filtration - the bound drug remains attached to the the filter
5) Count the radioactivity of the filter
6) Use a radiowave counter and analyse data
How can non-specific binding be reduced?
Using anti-absorbants eg albumin/ collagen for peptides and o-catechol for catecholamines
How can you discriminate between specific and non-specific binding in a radio ligand binding assay?
In one set of tubes there will be the tissue and the radiolabelled ligand - this shows specific and non-specific binding
In the second set there will be the tissue with radiolabelled ligand and an excess of unlabelled radioligand
The radiolabelled ligand has no chance to compete with the unlabelled ligand for binding with the specific site so the radiolabelled ligand is completely displaced to non-specific binding sites
What properties must the radioligand have for the radio ligand binding assay?
1) it must be biologically active
2) it must be purified
3) Shouldn’t degrade
How can degradation of a radioligand be prevented?
1) By using free radical scavengers eg ethanol in the drug solution
2) Store at a low, but not freezing, temperature
3) Avoid light by storing in a dark bottle
4) Incorporation of antioxidant (ascorbic acid)
What are the advantages of 3H as a radioactive label?
The labelled product is indistinguishable from native compound
High specific activities can be obtained
Good stability when properly stored
Long half life -12.5 years
What are the disadvantages of 3H as a radioactive label?
Specialised labs are needed
Labelling is expensive and difficult
What are the advantages of 125I as a radioactive label?
If the compound has an aromatic hydroxyl group it can be incorporated at very high specific activities
Iodination is easy in most labs and is cheap
What are the disadvantages of 125I as a radioactive label?
More readily degraded
Biological activity of ligand can be reduced
Short half life - 67 days
How is the tissue with the bound ligand separated from the free ligand remaining in incubation media?
Usually by filtration and centrifugation
But in soluble binding other techniques are used; dialysis, column chromatography and precipitation/adsorption
The speed of separation between the tissue and the free ligand must be compatible with what?
The affinity of the ligand for the receptor ie a lower affinity requires faster and more efficient separation
Does Kd increase or decrease if affinity increases?
It decreases (Kd is the dissociation constant so if affinity is higher less drug will dissociate)
At what point will non-specific binding become an issue?
When the concentration of the ligand rises above 1000um
What is the scatchard equation?
B/F = (Bmax - B) / Kd B = specific bound ligand F = free radioligand (approx concentration added) Bmax = max number of binding sites Kd = Equilibrium binding constant/ affinity
