Using the cultured cells used in this experiment and described in the introduction, how would you approach determining the potency of an agonist to the D2 receptor?

Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
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Chapter1: Biochemistry: An Evolving Science
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Using the cultured cells used in this experiment and described in the introduction, how would you approach determining the potency of an agonist to the D2 receptor?
Receptor binding can be assessed in a number of ways. One of
the simplest is to use a ligand that is radio labelled, usually with
³H or ¹4C; receptor binding can be calculated as a function of the
amount of radioactivity associated with receptors in a given
sample. With increasing concentrations of a radioligand, the
amount that binds to cells increases up to a point, then plateaus.
This represents total binding and includes binding of the
radioligand to receptors, together with other parts of the cell (e.g.
membrane phospholipids). In order to determine the level of
specific binding to receptors the assay must be repeated in the
presence of excess unlabelled ligand (either the same ligand or a
suitable competitor). Under these conditions, the radioligand
cannot bind the high-affinity receptor sites since these have been
occupied by the unlabelled ligand; the radioligands therefore
bind to non-receptor sites in a non-saturable fashion. This is
non-specific binding. The specific binding of radioligand to
receptor can be calculated as the difference between total and
non-specific binding.
In this data interpretation exercise, you are presented with real
data from a ligand binding experiment performed at the
University of Kent. This experiment assessed the reversible
binding to the D₂ dopamine receptor expressed in the membranes
of Chinese Hamster Ovary (CHO) cells. This will involve using
[³H]-spiperone, a dopamine receptor antagonist, in the presence
of (+)-butaclamol (for the analysis of non-specific binding) and
(-)- butaclamol (for the analysis of total binding). Data analysis
will be carried out to generate binding curves in order to
determine the K₂ and B value of the dopamine receptor
max
expressed in this cell line.
Transcribed Image Text:Receptor binding can be assessed in a number of ways. One of the simplest is to use a ligand that is radio labelled, usually with ³H or ¹4C; receptor binding can be calculated as a function of the amount of radioactivity associated with receptors in a given sample. With increasing concentrations of a radioligand, the amount that binds to cells increases up to a point, then plateaus. This represents total binding and includes binding of the radioligand to receptors, together with other parts of the cell (e.g. membrane phospholipids). In order to determine the level of specific binding to receptors the assay must be repeated in the presence of excess unlabelled ligand (either the same ligand or a suitable competitor). Under these conditions, the radioligand cannot bind the high-affinity receptor sites since these have been occupied by the unlabelled ligand; the radioligands therefore bind to non-receptor sites in a non-saturable fashion. This is non-specific binding. The specific binding of radioligand to receptor can be calculated as the difference between total and non-specific binding. In this data interpretation exercise, you are presented with real data from a ligand binding experiment performed at the University of Kent. This experiment assessed the reversible binding to the D₂ dopamine receptor expressed in the membranes of Chinese Hamster Ovary (CHO) cells. This will involve using [³H]-spiperone, a dopamine receptor antagonist, in the presence of (+)-butaclamol (for the analysis of non-specific binding) and (-)- butaclamol (for the analysis of total binding). Data analysis will be carried out to generate binding curves in order to determine the K₂ and B value of the dopamine receptor max expressed in this cell line.
Introduction
Hormone receptors bind ligands with high affinity using ionic,
van der Waals and hydrophobic interactions. This binding can
be represented as a simple reversible reaction.
Where R =
receptor, H free hormone and RH
receptor
hormone complex. Therefore the dissociation constant of the
receptor-ligand complex, K, can be defined as follows:
Fractional ligand binding [RH]/R+]
[RH]
R₁
0.5
R+H
where the affinity of the receptor for its ligand is inversely
proportional to the K, value. When written in the form of the
Michaelis Menten equation:
Total binding.
=
KD
RH
K₁ = [R][H]
[RH]
Where R = total number of free [R] and bound [RH] receptors,
and the K₂ value equals the concentration of ligand required to
occupy half of the total number of available receptors:
Dose-occupancy curve
Specific binding.
=
Non-specific binding
Ligand concentration
1
1 +K₂/[H]
Figure 1. Typical dose-response curves of a receptor binding
assay
Transcribed Image Text:Introduction Hormone receptors bind ligands with high affinity using ionic, van der Waals and hydrophobic interactions. This binding can be represented as a simple reversible reaction. Where R = receptor, H free hormone and RH receptor hormone complex. Therefore the dissociation constant of the receptor-ligand complex, K, can be defined as follows: Fractional ligand binding [RH]/R+] [RH] R₁ 0.5 R+H where the affinity of the receptor for its ligand is inversely proportional to the K, value. When written in the form of the Michaelis Menten equation: Total binding. = KD RH K₁ = [R][H] [RH] Where R = total number of free [R] and bound [RH] receptors, and the K₂ value equals the concentration of ligand required to occupy half of the total number of available receptors: Dose-occupancy curve Specific binding. = Non-specific binding Ligand concentration 1 1 +K₂/[H] Figure 1. Typical dose-response curves of a receptor binding assay
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