1. 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).

2. Ionotropic vs metabotropic/ overview

3. 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

4. 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.

5. 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

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

7. 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

8. 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.

9. 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

10. 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

11. 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.

12. 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 Tells something about where effective drugs probably act, and therefore what may be wrong with dopamine transmission in schizophrenia.

13. 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.

14. 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.

15. 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.

16. 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)

17. 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?

18. 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

19. 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.

20. Best studied in glutamate receptors and learning and memory.