Download felix may 2nd year neuroscience Neuroreceptor characterisation by

felix may
2nd year neuroscience
Neuroreceptor characterisation by radioligand binding
Abstract
The Muscarinic acetylcholine receptor is present in the autonomic nervous system. Atropine is a
chemical originally procured from atropa bellandonna and now synthesised for its antagonism of
muscarinic acetylcholine receptors. We used the radioligand [ 3H]-QNB (quinuclidinyl benzylate) - a high
affinity antagonist for all mACh rececptor subtypes - to determine the affinity of atropine for the mAch
receptors present in a sample of brain. Atropine was infused in concentrations ascending logarhymicly,
and the radioactive decay given off from each sample was measured. The decompositions per minute
indicate the quantity of QNB bound in the membrane. Because atropine and QNB will compete for
occupancy of receptors, the binding of atropine at the same sites could be deduced.
We found that atropine had an IC50 of 2.77 nM and a Kd of 1.385nM in this prodcedure, confirming that
it is a high affinity ligand for the mAch receptors we used in our tests.
Introduction
Observing the response to a receptor ligand in tissue or cells is useful in characterising the action of the
ligand on that receptor, and the function of the receptor itself. Muscle contraction or membrane
polarisation are typical examples of quantitive measures for drug action. Values like the EC50 and IC50
can be deduced, along with useful information on the substance and its efficacy. However in such tests
the underlying molecular pharmacology and biology of the responding system remains a black box.
Knowing the binding affinity of a ligand for a receptor, and its effect on tissue and cell preparations,
provides the key to finding the efficacy of a ligand.
Using an in vitro binding assay for a receptor allows some precise measurements of its affinity for
ligands. This is useful for screening compounds intended for therapeutic use, as well as characterising
existing ones.
Radioligand binding is a commonly used technique for assaying ligand affinity. A radiolabelled ligand is
applied to a membrane-receptor preparation, given time to equilibrate, an then washed to remove
unbound ligand. The remaining ligand is bound to receptor fixed fragments of membrane, which will not
pass through the filter. The filter paper is then analysed for radioactive deterioration, which is quantified
by a machine and adjusted against a calibration curve.
Membranes are sourced from lysed cells. Broken cell preparations can be natural in origin, expressing the
full host of receptors and surface proteins, or they can be engineered for a specific receptor. The
receptor’s DNA is isolated and cloned in a bacterial vector for expression in the sacrificial cells.
Muscarinic acetyl choline recetors are found in cardiac muscle, endothelium, and throughout the airways
and the gut, aswell as in the CNS. Outside of the CNS they modulate cardiac and smooth muscle
contraction. Within the CNS mAchr’s are found in the cerebral cortex, striatum, hippocampus, thalamus
and in the brainstem, inclusing the reticular formation.
Methods
Covered in detail in the schedule handed out at the beginning of the practical.
In brief:
We prepared atropine solutions in logarhythmic concentrations by serially diluting a stock solution.
Brain membrane samples were pre-prepared, containing about 3.5mg of protein per ml.
Membrane samples were infused with QNB, and then with varying concentrations of atropine, all done in
triplicate.
The membrane samples were individually filtered and the filter paper taken to measure the
decomposition of radioactive material being produced.
deviations and additions to the schelude:
The cell harvester was not working effectively, and we had to perform some of its functions by hand,
adding a source of variation and error to the process. In addition the large number of samples to be
filtered meant that some were left for longer to equlibrate, again adding inconsistency.
The radioligand binding assay is often assisted heavily by automated machinery. The samples are
prepared by a robot, the cell harvesters only require minimal human input, and of course the radioactive
decay measurement is performed by a machine. In our experiment many of the steps involved human
error and were not as consistent as they could have been because of this.
Results
figure 1 - sheet of results and manipulations
figure 2 - graph of calculated QNB concentration bound to mAch receptors against atropine
concentration
figure 3 - Hill plot of data with x-intercept at 8.573
figure 4 - graph of percentage atropine’s binding to mAch receptors against its concentration
values of interest:
[L] is 0.24nM
IC50 atropine = log-8.557 M = 2.77 nM
Kd = 0.24 * IC50 = (0.24 * 2.77)/0.48 = 1.385nM
0.24 + 0.24
from the hill plot IC50 atropine = log-8.573 = 2.67nM
hill slope = 1.359 (?!?)
50ul membrane samples, containing 3.5mg of protein per ml, will yield 0.175mg of protein per sample
Bmax = 1.132 e-8 M
total receptors in preperation = Rt = Bmax
Rt per mg of protein = Bmax/0.175 = 64.7 nM mg-1
Discussion
Binding of QNB can be seen to reach a baseline value where no additional atropine can displace it (fig.1).
This is considered to be ‘undisplaceable’ radioligand that is bound to sites other than mAch receptors, or
even just absorbed in the filter paper. Subtracting the lowest value for QNB binding yields the ‘specific’
binding of QNB. These figures can be considered the inverse binding of atropine - that is because if a
receptor is not occupied by one, it will be by the other. Binding of atropine to muscarinic acetylcholine
receptors (mAchr’s) follows the predicted trend of a sigmoidal curve when plotted against the
concentration of atropine added to the preperation (fig.4).
From figure 2 one can estimate a value for the IC50 of atropine. This is found as the molar concentration
that intersects the sigmoidal curve at 50% of maximum binding.
The IC50 value, like the EC50, gives a useful indication of a drug’s efficacy, that is how effective it is at a
given dose. A low IC50 signifies that a low concentration will produce 50% inhibition, so the lower the
IC50 is the more effective the drug is per mole. These values are used by pharmacologists extensively as a
guidline for effective dosing regimes.
Similarly the Kd value is of great interest in pharmacodynamics as it indicates the affinity of a ligand for
a receptor type or group of receptors. A low Kd indicates a high affinity of the drug for its receptor.
Drugs of high affinity are often therapeutically preferable, because less of the compound is required to
exert an effect, reducing the likeliness of side effects.
The hill slope indicates the type of interactions that are occuring at the receptor and molecular level. A
value below one indicates that the two substances are inhibiting each other’s ability to bind. A value
above one indicates the opposite, that each assists the other in binding, but this is rare. We were
expecting a value near 1 or just below. The relatively large hill slope value of 1.359 was a total shock… it
is hard to reconcile such a result. Perhaps this slope value was due to a calculation error.
Errors in the procedure itself, as described in the methods section, meant that out of the 7 or so sets of
data only 2 were of any use for analysis. Performing the experiment with groups, or with better assigned
time slots, would have created fewer bottle-necks in the procedure and thus created less error prone, more
useful results.
Our calculations and manipulations are resting upon certain assumtions. Firstly that receptors in the
membrane are available constantly and equally for binding, and secondly that binding is an all-or-none,
infinitely reversible process. Both of these assumptions are tenuous, but allowable in that their
implications will usually not intefere with some basic manipulations of the data.
Im summary our results conclude that atropine is a high affinity antagonist at mammalian brain
muscarinic acetylcholine receptors.