Apparent shut time 11 < tcrit (ms)

Apparent open time (ms)

Apparent shut time 11 < tcrit (ms)

Figure 11.14. Distributions of (logarithmsof)the apparent open time (left)and apparent shut time (right) for wild-type human receptors (top) and for mutant eL221F receptors (bottom).The histogram shows the experimental observations. The continuous lines were not fitted directly to the data in the histograms, but were calculated from the rate constants for the mechanism that was fitted (Fig. 11.10, scheme B, with the two sites constrained to be independent). The distributions were calculated with appropriate allowance for missed events (HJC distributions) (65, 66). The fact that they superimpose well on the histograms shows that the mechanism was a good description of the observations. The dashed lines show the distributions calculated from the fitted rate constants in the conventional way (45), without allowance for missed events, so they are our estimate of the true distributions cf open and shut times (296).See color insert.

described are consistent with those of macroscopic jumps, and simulations (with HJCFIT) show that this method can clearly give good estimates, at least of the main doubly liganded rate constants. For the method to give misleading results the reaction mechanisms on which it is based (like those in Fig. 11.12) would have to be in some way seriously wrong.

Obviously, it is to be expected that mutations in residues that form part of the physical binding site will affect agonist binding, and most of those discussed above do so. They mostly affect gating, too, but this is not unreasonable. Because the act of binding has to be transmitted to other parts of the molecule to trigger the large conformation change that occurs on opening, it is perhaps not at all surprising that residues such as Y190, which al most certainly form part of the binding site itself, also influence the gating process. It is harder to explain why «Y198F shows hardly any effect on binding, although according to the analogy with AChBP it is part of the binding site. On the other hand, the lack of effect of on binding, yet strong effect on gating, is consistent with its position some distance from the binding site, as predicted from the AChBP structure, but at the base of the C-loop where it could play a role in initiating the extended conformational change.

It is salutary, though, to notice that one of the purest binding effects is produced by the mutation, and it is unlikely that this residue is part of the binding site. Of course, it is usually easy to producepost hoc rationalizations of such results. A glycine to serine muta-

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