Receptor and Binding Studies Peter Hein, Martin C

723 6.3 Receptor and Binding Studies Peter Hein, Martin C. Michel, Kirsten Leineweber, Thomas Wieland, Nina Wettschureck and Stefan Offermanns 6.3.1 Radioligand Binding Studies in Cardiovascular Research (Saturation and Competition Binding Studies) Peter Hein and Martin C. Michel Introduction Radioligand binding studies are an important tool to quantify and qualitatively char- acterize receptors and hence have become a universal tool of cardiovascular research. While radioligand binding experiments are relatively simple to perform, interpreta- tion of the results and optimisation of the experimental settings are more complex. Several criteria must be met to consider a labelled binding site a receptor: Binding should be of high affinity, saturable, stereospecific and have a ligand recognition pro- file similar to that of a functional response in that tissue. A hilariously funny descrip- tion of the fundamental principles of radioligand binding has been presented by Guth (1982). Radioligand binding experiments can be divided into two major types of experiments, kinetic and equilibrium studies. Kinetic (i.e. association and dissociation) studies are required to define optimal assay conditions. On theoretical grounds they also allow the most reliable determination of the affinity of a radioligand for its cognate receptor. However, kinetic studies are cumbersome to perform and hence rarely used in routine studies. Their most frequent application is in the phase of characterization of novel radioligands. Once radioligands have been characterized and proper assay conditions have been established, the translation of such assays to cardiovascular tissues can be done in most cases without repeating the kinetic studies. The vast majority of applications of radioligand binding studies are based on equilibrium binding studies. The key molecular principle underlying such experiments is the idea that association of a radioligand with its cognate receptor and dissociation of the resulting ligand-receptor complex are reversible processes that occur concomitantly until equilibrium has been reached. Key factors in determining the 6 724 Biochemical and Analytical Techniques Figure 1 A saturation binding experiment allows measuring Kd and Bmax of a ligand/receptor-pair development of equilibrium conditions are incubation time, incubation temperature and assay volume (the latter as a measure of amount of radioligand relative to number of binding sites in the assay). Equilibrium binding studies can be divided into two main types of experiments, i.e. saturation and competition studies (with the latter often but incorrectly referred to as displacement studies). Saturation binding studies In-Vitro Techniques allow determination of the affinity of a receptor for a radioligand and, more impor- tantly, receptor densities. Competition binding studies allow a pharmacological characterization of receptors and, under proper experimental conditions, identification of the receptor subtype distribution within a tissue or cell line. Saturation binding studies are performed using several concentrations of a radioligand and allow determination of the dissociation constant (Kd) and number of binding sites in the assay (Bmax) for the radioligand (Fig. 1). The Kd-value is a measure of the affinity of the ligand for its binding sites. Since the Kd is the concentration at which the ligand binds to half its binding sites, the ligand’s affinity for the receptor is small when its Kd is large. Likewise, ligands with a high affinity have small Kds. Some- times the affinity is given as pKd, i.e. the negative logarithm of Kd. Kd is a constant and, unless a new radioligand or receptor is characterized, is largely used as an internal quality control of a binding assay. The receptor density in a tissue or cell line is called Bmax. Bmax values are typically adjusted to account for tissue content in the assay, e.g. for protein content or tissue wet weight and expressed as fmol/mg protein or fmol/mg wet weight, respectively. Saturation binding experiments can also be used to determine whether a given drug indeed acts as a competitive antagonist at a receptor of interest. Thus, concomitant saturation binding experiments in the absence and pres- ence of a competitor should yield identical Bmax values but distinct apparent Kd values if the competing drug acts in a competitive manner. In this case the shift in Kd value can even be used to determine antagonist affinity in a manner analogous to that used when analyzing the shift of an agonist concentration-response curve by an antagonist in organ bath experiments with isolated tissues. Receptor and Binding Studies 725 6.3 Figure 2 C50, the concentration of the ligand causing 50% inhibition of radioligand binding, is de- termined by a competition binding experiment Competition binding studies, sometimes also referred to as displacement studies, use a single concentration of a radioligand that is competed for by multiple concentrations of another, not radioactively labelled compound (Fig. 2). They allow the pharmacological characterization of binding sites. Competition studies primarily yield the concentration of the inhibitor causing 50% inhibition of radioligand binding (IC50), which can be converted into the inhibitor constant of the inhibitor (Ki) using appro- priate equations. If a tissue contains multiple binding sites for a given radioligand and the competing compound differentiates them, biphasic or even multiphasic competition curves are obtained which allow not only determination of Ki values at the vari- ous sites but also an estimate of the relative quantitative contributions of each site. The following considerations are primarily developed for radioligand binding studies with G-protein-coupled receptors, which are the most frequent subjects of studies in cardiovascular research. While radioligand binding studies to other receptor classes such as ligand-gated ion channels, receptors with intrinsic enzyme activ- ity or ligand-activated transcription factors (e.g. glucocorticoid receptors) follow similar principles, not all details given below apply to these other receptor classes. On the other hand, many of the approaches described hereafter can also be applied to non-receptor proteins such as uptake carriers using minor variations of the protocols given below. Descriptions of the Methods and Practical Approach Receptor Theory Binding of the ligand to the receptor follows the law of mass action. The ligand and receptor associate and form a ligand-receptor-complex, or they may dissociate again. 6 726 Biochemical and Analytical Techniques receptor + ligand ↔ receptor · ligand (1) The association rate is called Kon, and the amount of newly associated receptor-ligand complex depends on Kon, the concentration of the free ligand and the concentration of free receptors. Accordingly, the dissociation rate Koff, and the concentration of ligand- receptor complexes describe the number of dissociations. At equilibrium concentrations, the rate at which new receptor-ligand complexes are formed equals the rate at which they dissociate. × × × [ligand] [receptor] Kon = [receptor · ligand] Koff (2) This may be rearranged to [ligand] × [receptor] K = off = K (3) ⋅ d [receptor ligand] Kon The quotient Koff/Kon is named the equilibrium dissociation constant Kd. It has the units of a concentration and, more specifically, is the concentration at which half the receptors are occupied by the ligand or free, respectively. Note that there is a specific Kd value for each ligand/receptor-pair, and since in different tissues different receptor- subtypes (or even conformational different states of the same subtype) may be present, there may be different Kd values for the same ligand and receptor in different tissues. In-Vitro Techniques Koff has a major effect on the time required to reach equilibrium and hence usually governs the choice of incubation time for saturation and competition equilibrium binding studies. Equilibrium is often reached after 45–60 min, but with some ligands or receptors, longer time periods may be necessary. It should also be noted that Koff is affected by the incubation temperature, but this has little effect on kinetic determination of Kd as long as Kon and Koff are measured at the same temperature (for criteria for choice of temperature see below). Radioligands Chemically, radioligands can be derived from agonists or antagonists, and it should be noted that the two classes of drugs label different receptor populations. Thus, antagonists theoretically should label all receptors while agonists typically only label a frac- tion thereof with high affinity. Therefore, antagonists are usually preferred as radioligand unless labelling the receptor population in a high affinity state for agonists is specifically attempted. However, for some receptor classes, e.g. for many pep- tide receptors, only agonist radioligands are available, which are typically radio- labelled forms of the endogenous ligand. Moreover, it should be noted that in some cases antagonist radioligands from distinct chemical classes may label different num- bers of receptors, even if it is guaranteed that only one receptor subtype exists within the assay (Hein et al. 2000). Receptor and Binding Studies 727 6.3 In some cases a given receptor can be labelled with either a more lipophilic or a more hydrophilic radioligand, e.g. for muscarinic acetylcholine receptors with [3H]-quinuclidinyl-benzylate or [3H]-N-methyl-scopolamine,

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