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

Abstract

British Journal of Pharmacology (1998) 125, 923–947; doi:10.1038/sj.bjp.0702103 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 affinity for the binding of the agonist has also been reduced by about 100 fold. Obviously the mutation has affected 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 affinity for the binding step of the reaction was totally unaffected by the mutation (the equilibrium constant for this step was 100 μM for both wild type and mutant). The only difference 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 affect the ability to change conformation should be anywhere near the agonist binding site. Concentration-response curves (left) and agonist-binding curves (right). Calculated from the del Castillo-Katz (Scheme 1). The binding reaction has an equilibrium constant of KA= 100 μM for both wild type and mutant receptors, so the mutation does not affect the binding site at all. The equilibrium constant for isomerisation to the open state (the gating reaction) is 200 for the wild type (high efficacy), but only 1 for the mutant. The mutation has affected only the ability of the protein to change its conformation; the binding site is unaffected. 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 effects on binding and effects 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 efficacy 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 structure-activity relations and of mutant studies, that efficacy has no upper limit in principle, and that when it is large, changes in it are indistinguishable from changes in affinity. (b)‘This change in the sensitivity of the Y190F mutant could be due either to a change in the binding affinity of the receptor or to a change in the energetics of opening of the channel after binding. To distinguish between these possibilities, we assessed the ACh binding affinity of the mutants by measuring the ability of ACh to compete for [125I]-α-bungarotoxin binding’ (Tomaselli et al., 1991). Figure 1 shows clearly that such measurements do not make the required distinction. (c)‘Of particular interest in this regard is the ability to measure independently both ligand receptor affinity (by ligand binding techniques) and biological activity (ED50 and Emax). These two independent processes provide information about …’ (Hollenberg, 1991). The whole point of the problem is, of course, that these measures are far from being independent. If binding affects activation (transduction, gating), then activation must affect binding. (d)‘Measurement of full-agonist-affinity can be made by a procedure developed by Furchgott’ Kenakin (1997a, p272). This would be true only if activation had no effect on binding, which is not true for any currently proposed mechanism of agonist action (Colquhoun, 1987). The problem of interpreting the effect of mutations has been discussed before (Colquhoun & Farrant, 1993). It now seems timely to consider what can be done about it. Although difficult, it is not impossible, and, at least for ion channels, there are sensible things that can be done. Before going on to the modern problem, it is appropriate to recognise that this is not a new problem at all. Essentially the same difficulty occurs in the interpretation of the structure-activity relationships of a series of different agonists on the same receptor, and in this context it has been around since the 1950s. Stephenson (1956) had pointed out the very important fact that the action of an agonist could not be described by an affinity constant alone. In