Neuroreceptor characterisation by radioligand binding
Abstract
A displacement radioligand assay was used to measure the number of atropine
binding sites and the Kd of atropine.
Introduction
Traditional approaches in receptor experimentation provided sufficient information to
construct a general theory for receptor function by investigating the responses of
effector cells to transmitters in intact preparations. However these methods did not
allow for the measurement of receptor concentrations or analysis of the chemical
nature of receptors and so a method to use broken cell preparations was developed.
Radioligand binding works on the basis that if you radiolabel a drug that binds to a
receptor you can measure the amount of radioactivity that is associated with the
receptor to deduce drug affinity (KD) and the number of receptors in the tissue.
Affinity is the tendancy for a ligand to bind to a receptor.
The degree of binding depends upon the concentration of drug and receptor as well as
the affinity between them and is governed by simple mass action law. This law states
that equilibrium is reached when the rate at which new ligandreceptor complexes are
formed equals the rate at which the ligandreceptor complexes dissociate. This is
represented by this formulae:
[ligand] [receptor] . Kon = [ligand . receptor] . Koff
For this assay a fixed concentration of labelled ligand has to compete with an everincreasing concentration of labelled ligand with the model assuming that the binding
is reversible. This is known as a displacement assay.
In this experiment we are going to investigate the presence of muscarinic receptors.
We are using two muscarinic antagonists, QNB (quinuclidinyl benzylate) as the
radioactive ligand and atropine to displace it. Antagonists are chemical substances
that interfere with the physiological action of the receptor by combining with and
blocking it.
All parasympathetic postganglionic axons release acetylcholine (ACh) the effects of
which are brought about by the action of muscarinic cholinergic receptors (mACh-R).
Compared to nicotinic receptors, which are also responsive to ACh, muscarinic
responses are much slower. Five different types of muscarinic receptors have been
found so far and all but one type are coupled via G proteins to adenylyl cyclase, K+
channels or phospholipase C. The effects produced by this intracellular cascade
system may be either excitatory or inhibitory and do not involve the direct opening of
an ion channel.
The major tissues apart, from autonomic ganglia, containing mACh-R are cardiac
muscle, exocrine glands and endothelium as well as gut and airway smooth muscle.
They act to regulate cardiac contractions, gut motility and bronchial constriction.
They are also found throughout the brain, including the cerebral cortex, the striatum,
the hippocampus, thalamus and brainstem.
The aims of this experiment are to investigate a sample of mammalian brain
membrane in order to determine the number of muscarinic receptors and the KD of
atropine to order to work out the affinity of the membrane for atropine.
Methods
The experiment was carried out as instructed in the handout. 50 l of membrane was
added to different concentrations of atropine ranging from 10-11 to 10-4 and 900 l of
QNB. These were incubated for 30 minutes at 37c before being filtered using a Cell
Harvester.
We encountered many problems whilst working through the protocol. The first was
met during the stage when the brain membrane was added to the vials. There was a
delay between putting the membrane into the first vial and the later vials, which may
have caused prolonged binding reactions in the first vials. It has been given more time
to reach equilibrium and so the balance will be different than for later vials.
There is also the usual problem during experiments of human error and equipment
inaccuracy. Not all the measurements may be have precise due to tiny amounts of
solution being left in the pipettes and vials.
Due to the large size of the group it was necessary to stagger the start times of
incubation and a backlog led to some vials staying in longer than the allotted
incubation time which may have altered the binding equilibrium.
A major difficulty was with the Cell Harvester which encountered a technical problem
due to earthing. The buffer to terminate, wash and filter the samples had to applied by
hand using a spray bottle. This may have resulted in improperly filtered samples and
free radioligands being bound up in the filter paper and counted in total amount
bound.
Ensuring the Cell Harvester was working correctly, a quicker system for the
membrane addition and making sure that the samples were all terminated at the same
time could have reduced these errors.
Other errors that occur are due to the fact that as radiolabelled ligands are so
expensive we only used the Kd concentration but using a lower concentration results
in fewer counts of specific binding and thus more counting error.
Results
Figure 1. Table to show the average disintegrations and the amount of QNB
bound for varying concentrations of atropine
Concentration of atropine Average
disintegrations Amount of QNB (pmoles)
(moles)
per minute of QNB (dpm)
10-11
5361.25
0.056
10-10
5101.69
0.053
10-9
3707.04
0.039
10-8
1858.55
0.019
10-7
487.39
0.0051
10-6
256.29
0.0027
10-5
239.37
0.0025
10-4
234.78
0.0025
Figure 2.
Amount of bound QNB
(pmoles)
Amount of QNB bound to mACh-R for varying
concentrations of atropine
0.06
0.05
0.04
0.03
0.02
0.01
0
11
10
9
8
7
6
5
4
-log[atropine] (M)
Figure 3. Calculation of Kd of atropine using the estimated IC50 value
Reading from the graph above the IC50 = log (-8.54)
Concentration of atropine = 3.16 x 10-9 moles
As [KL] = [L] and Kd for QNB is 0.24 nM
KD = (0.24 * 3.16 x 10-9 ) / (0.24 + 0.24)
KD = 1.5 x 10-9 moles
KD = 1.5 nM
Figure 4. Percentage of QNB and atropine bound worked out by taking away the
non-specific binding to leave the specific binding of the QNB
Concentration Amount
of
atropine QNB
(moles)
remaining
bound
(pmoles)
of Specific
Percentage of Percentage of
binding
of QNB bound
atropine bound
QNB (pmoles)
(b)
10-11
0.056
0.0535
100
0
10-10
0.053
0.0505
94.4
5.6
10-9
0.039
0.0365
68.2
31.8
10-8
0.019
0.0165
30.8
69.2
10-7
0.0051
0.0026
4.9
95.1
10-6
0.0027
0.0002
0.37
99.63
10-5
0.0026
0.0001
0.1
99.9
10-4
0.0025
0
0
100
Figure 5.
Hill plot for varying atropine
concentrations
2
log b/B-b
1
0
-1
-2
-3
-4
0.0
2.5
5.0
7.5
10.0
12.5
-log [atropine] pmoles
Figure 6. Calculation of new Kd for atropine using IC50 derived from the Hill
plot
From figure 5 the new IC50 = log (-8.172)
Concentration of atropine = 6 x 10-9
KD = 3 x 10-9 moles
KD = 3 nM
The slope of the Hill plot = 0.1359
Figure 7. calculation of the total number of atropine binding sites
There is 3.5mg of protein per ml of fluid
Therefore there is 175 g of protein in 50l of incubation fluid.
conc of protein is worked out via the methods values, use 3.5mg/ml, we used 0.050ml
so worked out ratio of 20,
then ratiod down the 3.5ml/mg, got 0.175mg in the 0.050ml of brain membrane we
used
worked out QNB displaced / mg protein in samples,
0.053x10-9 / 0.175x10-3
=3.03 x 10-7 pmol/mg
ive got a value of 35.5pmol / mg protein of receptor
The total number of atropine binding sites:
There is 3.5mg of protein per ml of fluid thus there is 175 ug of protein in 50ul of incubation fluid.
At 50% occupancy KD was 1.58
By using the equation: KD= free receptors *[receptors]
/
Bound receptors
At KD there is 50% occupancy, at this point the number of free receptors and bound receptors are
equal (as they are both 50%) so the values can cancel out.
1.58= 50% free*[receptors]
/
50% bound
Cancel out (they are the same)
1.58= [receptors] (in 50ul)
Also using the equation:
[Total receptors]= [receptor] + [receptor-ligand complex]
The [total receptor] can be worked out as the occupancy is a 50% the [receptor] and [receptor-ligand
complex] are equal.
Thus:
[Total receptors]= [1.58] + [receptor-ligand complex]
[Total receptors]= [1.58] + [1.58]
[Total receptors]= 3.06 (in50ul)
To find the total number of receptors in an ml, multiply the number by 20
3.06*20 = 61.2 receptor binding sites per ml
Discussion
From figure 2 it was possible to determine a rough estimate of the IC50 value. An
IC50 value is the concentration of unlabeled drug that blocks 50% of the specific
binding of the radiolabelled ligand.
As can be seen on the graph (figure 2) the bottom of the curve does not go down to
zero but runs along just above it. This is due to non-displaceable binding which
occurs because some of the QNB is bound to non-specific sites on the membranes or
may be stuck in the filter and cannot be displaced by the atropine. This means that
you never get the maximum occupancy of QNB.
Specific binding is when the QNB is bound only to muscarinic receptors and can be
calculated out using the information from figure 2. The values used to plot that graph
are those for the non-specific binding of the QNB including results recorded from
non-displaceable binding of QNB. To work out specific it is necessary to minus the
lowest value, the non-displaceable binding, from all the other values giving a baseline
value of zero.
The first KD value worked out from figure 2 was 1.5 nM and from the Hill plot in
figure 5 KD came out as 3 nM. Typical values for KDs are between 10 pM and 100 nM
and show our results are within reasonable boundaries. The difference in KD values
might be because the Hill plot is double-logarithmic plot and so allows more accurate
readings to be taken and used in formulae. The equilibrium constant KD is a
characteristic of the drug and the receptor and represents the affinity of that drug for
the receptor. Our KD is fairly low and so suggests that atropine has a high affinity for
binding to the muscarinic receptors.
In figure 6 the slope of the hill plot was recorded as 0.1359. As this is a value less
than one is shows negative cooperativity. A molecule has cooperative binding if when
binding a ligand the affinity of the ligand for the molecule changes depending on the
amount of ligand already bound. If a molecule has negative cooperativity as more
ligand is bound the affinity for the ligand decreases.
Typical values might be a Bmax of 10-1000 fmol binding sites per milligram of
protein
This experiment is based upon several assumptions such as all receptors are equally
accessible to ligands, all receptors are either free or bound to ligand and ignores the
possibility of partial binding, that neither the ligand or the receptor are altered by
binding and the binding is reversible. The accuracy of the value for total atropine
binding sites depends upon how much these assumptions apply to the muscarinic
receptors and atropine ligand.
Knowledge of muscarinic receptors has been useful in the understanding of diseases
such as Alzheimer’s and Parkinson’s. Alzheimer’s disease is associated with a loss of
cholinergic neurons, using muscarinic receptors, which project from the basal
forebrain to the cerebral cortex and the hippocampus. The loss of cholinergic neurons
is progressive and results in profound memory disturbances and irreversible
impairment of cognitive function. Recent efforts have focused on the development of
centrally active muscarinic receptor agonists for the treatment of Alzheimer's disease.
Radioligand analysis allows the properties of such a drug to be calculated, such as its
affinity and cooperativity.