1. Outline the main reasons for undertaking a radioligand bindingexperiment? Ligand bindingexperiments are carried out to investigate binding of a ligand to receptors in order to create therapeutic drugs thatare selective, or mimic endogenous ligands. Identifying the rate and extent of ligand binding provides information onbinding sites, their affinity and accessibility for a range of drugs (Motulsky,1995-96). A radio-ligand is a drug with a radioactive label that can associate to anyprotein of interest (Motulsky, 1995-96). Radio-ligand assays are commonly seenin the screening process of drug development where the affinity of a new drugfor a range of receptors is determined and quantified.2. Name two basic types of binding experiments and list the bindingparameters that can be determined from each type of experiment. Saturation binding experimentsmeasure the specific binding at equilibrium of various radio-ligandconcentrations and determines receptor number and affinity.
Saturationexperiments allow for the determination of the dissociation constant (Kd)andthe receptor density measure (Bmax). Competition binding experiments use a single concentration of radio-ligand inthe presence of various concentrations of unlabelled ligand (Motulsky,1995-96). Competitive binding experiments yield an IC50 (inhibitoryconcentration 50%) from which the Ki can be derived by using the Cheng andPrusoff equation.
3. How are 5-HT receptors classified and where are they found in thebody? 5- HT receptors are asuperfamily (7 in total) of G-Protein Coupled Receptors and ligand-gated ionchannels found throughout the central and peripheral nervous system, where theymediate both excitatory and inhibitory neurotransmission (Rang, et al., 2016). Receptor classification is based on pharmacological response and this can beachieved through a number of ways; direct measurement of ligand binding(agonists and antagonists) to receptor, molecular cloning, and through theanalysis of transduction mechanisms/ biochemical pathways and second messengersthat are linked to receptor activation (Hoyer, et al.
, 1994) (Rang, et al.,2016). 4.
Explain why agonist affinity cannot be measured by saturationbinding.Agonistbinding to a receptor causes a conformational change to occur which leads to achange in affinity; affinity is a constant and thus the measurement following aconformational change would be inaccurate. Assuming the law of mass action maynot be true when the ligand is an agonist, antagonists are generally used inbinding experiments.