Lecture 7

Question 1

What are the binding interaction between noradrenaline and beta2 adrenoceptor?

Answer 1

• Amino acid residues in transmembrane 3, transmembrane 5 and transmembrane 6 interact with different parts of the endogenous ligands, noradrenaline:
  ○ hydroxyl group interact with serines in transmembrane 5
  ○ beta hydroxy group interacts with asparagine in transmembrane 6
  ○ phenolic ring interacts with a phenylalanine group within transmembrane 6
• quaternary amino group interacts with an aspartate within transmembrane3

Question 2

Define Kd

Answer 2

Concentration of ligand required to bind 50% of available sites at equilibrium

Question 3

Define Law of Mass Action

Answer 3

Equilibrium is reached when the rate of formation of new receptor-ligand complex is equal to the rate of dissociation, so that there is no net gain in ligand-bound sites

Question 4

What are the ways to measure ligand binding?

Answer 4

• Radioactive labelled ligands:
  ○ Addition of radioactive atom (e.g. tritium) to ligand to make it radioactive
  ○ Incubation of cells with radioligand until equilibrium is reached
  ○ Then separate receptor-bound ligand and free ligand via filtering them through filter paper
  ○ Free ligand is filtered out whilst the receptor bound ligand remains on filter paper
  ○ Measure them for radioactivity via adding an organic scintillant and monitor the flashes of light produced due to radioactivity.
  OR
  ○ Scintillant coated bead attached to receptor
  ○ Radioligand come in close contact with the receptor attached to the bead
  ○ Bead becomes excited and emit lights that can be detected and monitored using an instrument.
  
  • Fluorescent polarisation
    ○ Add fluorophore (e.g. Bodipy) to ligand
    ○ Laser light used to exicite the fluorophore when bounded to receptor to emit light which is detected by instrument
    OR
    ○ Can look at polarisation of light emitted from fluorophore attached to ligand
    ○ Unpolarised light emitted if ligand is unbounded to receptor i.e. free ligand
    ○ Polarised light emitted if ligand is bounded to receptor
    ○ Polarised light can be detected by an instrument
  • Use of other screening technology to measure light emitted from fluorophore
    ○ Direct visualisation: green membrane due to light emitted from the fluorophore
    ○ BRET
    ○ FRET
    TR-FRET

Question 5

Define dissociation rate constant

Answer 5

Off-rate is the fraction of ligand that dissociate in a given time so has only units of times

It is independent of the number of receptor bound at the beginning of the experiment

An off rate of 0.01 min-1 means that 1% of the bound ligand will dissociate in 1 minutes

Question 6

What is the residence time

Answer 6

Residence time is taken as the reciprocal of Koff. Eg. an off rate of 0.001min-1 gives a residence time of 100min.

Question 7

How can dissociation rate constant be measured?

Answer 7

• Incubate the membrane with radioligand or fluorescent ligand (N-methylscapolamine) till equilibrium is reached
  
  • Wash away radioactive ligand with infinite dilution of >100-fold (via adding buffer to membrane to dilute the membrane) or addition of high concentration of cold competitor (in this case atropine to prevent the re-binding of N-methylscapolamine to muscarinic receptor).
    This is to prevent the re-association of ligand to membrane receptor.
    Plot results on graph and find the half life of the ligand
    Positive allosteric modulator can increase residence time wheras negative allosteric modulator decreases it.

Question 8

What does the observed on-rate depend on?

Answer 8

on-rate, ligand concentration and off rate

Question 9

What to measure association constant?

Answer 9

Look at lecture 7 slide 112

Question 10

How to quantify competitive binding?

Answer 10

• Add a fixed concentration of radiolabel or fluorescent label ligand
  
  • Then, gradually increase the concentration of non-radiolabel or non-fluorescent competitive antagonist
    The competitive antagonist will compete for the same receptor, causing a gradual decrease in the binding of radiolabel or fluorescent label ligand to specific receptor hence reducing the amount of specific binding.

Question 11

Describe the equilibrium in competitive antagonist binding

Answer 11

○ Fluorescent or radiolabelled ligand binding to receptor to form ligand receptor complex.
○ Competitive antagonist binding to the same receptor to form competitor receptor complex
Addition of more competitive antagonist will lead to formation of more competitor receptor complex hence reducing the formation of ligand receptor formation.

Question 12

Describe Cheng Prusoff equation.

Answer 12

See last slide of lecture 7

Question 13

What determine the IC50 value of the competitive antagonist

Answer 13

concentration and affinity of the labelled compound used

Question 14

what are the choice of NSB-defining competitor

Question 15

Whar are the choice of NSB concentration