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 ofthe simplest is to use a ligand that is radio labelled, usually with³H or ¹4C; receptor binding can be calculated as a function of theamount of radioactivity associated with receptors in a givensample. With increasing concentrations of a radioligand, theamount that binds to cells increases up to a point, then plateaus.This represents total binding and includes binding of theradioligand to receptors, together with other parts of the cell (e.g.membrane phospholipids). In order to determine the level ofspecific binding to receptors the assay must be repeated in thepresence of excess unlabelled ligand (either the same ligand or asuitable competitor). Under these conditions, the radioligandcannot bind the high-affinity receptor sites since these have beenoccupied by the unlabelled ligand; the radioligands thereforebind to non-receptor sites in a non-saturable fashion. This isnon-specific binding. The specific binding of radioligand toreceptor can be calculated as the difference between total andnon-specific binding.In this data interpretation exercise, you are presented with realdata from a ligand binding experiment performed at theUniversity of Kent. This experiment assessed the reversiblebinding to the D₂ dopamine receptor expressed in the membranesof Chinese Hamster Ovary (CHO) cells. This will involve using[³H]-spiperone, a dopamine receptor antagonist, in the presenceof (+)-butaclamol (for the analysis of non-specific binding) and(-)- butaclamol (for the analysis of total binding). Data analysiswill be carried out to generate binding curves in order todetermine the K₂ and B value of the dopamine receptormaxexpressed in this cell line. IntroductionHormone receptors bind ligands with high affinity using ionic,van der Waals and hydrophobic interactions. This binding canbe represented as a simple reversible reaction.Where R =receptor, H free hormone and RHreceptorhormone complex. Therefore the dissociation constant of thereceptor-ligand complex, K, can be defined as follows:Fractional ligand binding [RH]/R+][RH]R₁0.5R+Hwhere the affinity of the receptor for its ligand is inverselyproportional to the K, value. When written in the form of theMichaelis Menten equation:Total binding.=KDRHK₁ = [R][H][RH]Where R = total number of free [R] and bound [RH] receptors,and the K₂ value equals the concentration of ligand required tooccupy half of the total number of available receptors:Dose-occupancy curveSpecific binding.=Non-specific bindingLigand concentration11 +K₂/[H]Figure 1. Typical dose-response curves of a receptor bindingassay

Question

Accepted Answer

When increasing amounts of the labelled ligand are added to the cells, the amount of binding will…