Receptive substances proposed 1909 by Langley (nicotine) Receptive substances proposed 1909 by Langley (nicotine). Before definitive demonstration of chemical transmission 1921 (Loewi). Two main classes of receptor; ionotropic and metabotropic mediate fast and slow transmission, respectively. All these found in the plasma membrane (steroid hormone receptors are intracellular, nuclear).

Ionotropic vs metabotropic/ overview

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. Knock out receptor get no response

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 1971. Overcome difficulty of making in vivo measures of receptor binding—drugs cross BBB, hydrophobicity of ligand may restrict access to some membranes, metabolism of drug before reaching receptor, compensatory changes may take place rapidly in the prolonged presence of the drug.

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 Become the criteria used today

Saturation binding Saturability and specificity. Specific=low capacity, high affinity

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 kD is equal to the concentration of ligand that produces 50% of maximal binding

Saturation binding curves QNB is an antagonist at the muscarinic acetylcholine receptor. Chemical warfare agent. Specific binding in different tissues. Point of plateau is Bmax; read concentration off Y axis. kD is the concentration of ligand required to reach half maximal binding.

Scatchard Transformation Not always easy to read Bmax and kD from a saturation plot. Plot bound/free vs bound (pmol/mg protein). Bmax is the x-intercept, kD is 1/slope. Steeper slope=higher affinity

Comparing slopes reveals that affinity does not change—probably the same receptor. But different x-intercepts, means different amounts of the receptor in different tissues

Displacement or indirect binding Displacement binding. At 50% displacement, concentration of inhibitor is the IC50. Related to ki formally, but usually we can assume IC50=ki. Displacement binding tells you whether two substances interact with (bind) the same receptor.

IC50 vs. Potency of different DA receptor antagonists Biologic potency of drugs that treat schizophrenia correlates with their ability to displace the dopamine receptor ligand haloperidol but not SCH-23390. Tells something about where effective drugs probably act, and therefore what may be wrong with dopamine transmission in schizophrenia.

Muscle relaxants and their ability to displace diazepam (valium) Correlation between the muscle relaxing properties of various drugs and their ability to displace diazepam (aka valium). Note that the best you can do is get a good correlation. Binding in vitro can not tell you about drug actions.

Hill Plot The slope is 1 when the receptor occupancy exactly corresponds to the number of binding sites. The response is equal to the amount of receptor bound. i.e. one binding site per receptor. Ex. Naloxone, an opioid antagonist.

Hill Plot-Cooperativity When the slope is not =1 or the line is not straight, it indicates cooperativity. Ie. There are multiple binding sites and the binding of one molecule influences the binding at other sites. These are allosteric influences. Not all allosteric interactions have to be the same molecule binding. Hemoglobin-4 O2 binding sites. First 02 bound makes subsequent affinity for O2 higher. CO, cyanide, NO and other toxic gasses can bind this site with higher affinity than O2, leading to death.

Some problems 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)

Explain: Caveats of radioligand binding assays in homogenized tissue…binding does not always correlated with levels of mRNA or protein. What else could affect binding?

Receptor localization Receptors must be found at post-synaptic density/membrane Complex systems of scaffolding molecules are in place to hold receptors at the right location Link receptors to downstream signaling pathways too

Receptor localization to plasma membrane—lipid rafts Receptor localization to plasma membrane—lipid rafts?—is a major determinant of binding capacity as well as signaling capacity. Scaffolding proteins serve to anchor receptors at the membrane as well as recruit and stabilize key downstream signaling molecules.

Best studied in glutamate receptors and learning and memory.