Transcript ppt
What are the criteria for a receptor? • Capacity to bind a ligand (not unique) • Membrane associated; induce biological response when bound (not unique) • Do not change the ligand by binding (not unique) • True neurotransmitter receptors do all the above and mediate the response to the transmitter. Radioligand binding assay • • • • Tissue homogenate High activity radioactive labeled ligand Unlabeled ligand Total binding measured in the presence of increasing concentrations of labeled ligand • Non-specific binding measured as above but in the presence of excess unlabeled (cold) ligand • Specific binding is the subtraction of the two Criteria defined by radioactive binding assays: A Specific Receptor • Saturability – Very small amount of receptor in tissue • Specific and high affinity – Found in biologically relevant sites – High receptor occupancy with low, physiologic concentrations of ligand • Biologic relevance – Distribution of receptor matches distribution of ligand – Binding or displacement of drugs should correlate with the potency of those drugs • Reversibility – Timing and recovery of ligand unchanged Binding characteristics • Bmax: density of receptors • kD: 1/affinity of receptor for the ligand • Binding according to law of mass action [L] + [R] [LR] Rate of reaction proportional to [ ] reactants K1=[L]*[R] K2=[LR] At equilibrium, k1=k2. Substituting, k1/k2=[L]*[R]/[LR]=kD, the equilibrium dissociation constant Scatchard Transformation Displacement or indirect binding Hill Plot Hill Plot-Cooperativity Some issues with radioligand binding data • Maximal response can be achieved with only a portion of receptors bound – Spare receptors • Agents that bind equally may yield different biological response – Full agonist, partial agonist, antagonist – Efficacy or intrinsic activity • Desensitization: decreased response with continuous exposure to drug (binding unchanged) Two state model • Receptors go between active and inactive state – Agonists prefer active state – Efficacy (response) determined by state preference (ratio of affinity for two states) – Antagonists prefer inactive state (no efficacy)