Binding, gating, affinity and efficacy: The interpretation of structure‐activity relationships for agonists and of the effects of mutating receptors

The nature of the problem is illustrated by the curves in Figure 1. A mutation in a receptor is seen to produce 100 fold increase in the EC50 for an agonist (Figure 1a). A ligand binding experiment with the same agonist, on the same mutant receptor (Figure 1b) shows that the measured anity for the binding of the agonist has also been reduced by about 100 fold. Obviously the mutation has a€ected the agonist-binding site, and the mutated amino acid is likely to be part of that site? No! It is not in the least obvious. The example in Figure 1 was calculated on the basis that the anity for the binding step of the reaction was totally una€ected by the mutation (the equilibrium constant for this step was 100 mM for both wild type and mutant). The only di€erence between wild type and mutant receptor in this example is the ability of the receptor, once the agonist has bound, to change conformation to its active state. There is no reason at all why the amino acids that a€ect the ability to change conformation should be anywhere near the agonist binding site. . Binding experiments do not measure affinity (in any sense that is useful for learning about the binding site), for any ligand that causes a conformation change. . The term `apparent affinity' is often used to describe EC50 for the response but it is meaningless (unless you define what you mean by `apparent'). Making this distinction between e€ects on binding and e€ects on conformation change is arguably the fundamental problem of modern molecular studies of receptors. It is not an easy distinction to make, but unless it can be solved, the interpretation of structure-function studies is quite likely to be nonsense. It is not just a theoretical problem; this is how ion channels actually behave. Nevertheless, the very existence of the problem has not always been recognized. For example, statements like the following are not at all uncommon*. (a) `Simplistically, the ecacy of a full agonist can be set equal to 1, that of an antagonist to 0, and that of a partial agonist to a value between 0 and 1' (Ross, 1996, in Goodman & Gilman, 9th Edition). This statement obscures the point that is crucial, both for the interpretation of structureactivity relations and of mutant studies, that ecacy has no upper limit in principle, and that when it is large, changes in it are indistinguishable from changes in anity.