Much evidence has suggested that the ACh binding sites are close to subunit interfaces, and the AChBP structure seems to confirm this view. Most of the residues of the a subunit that were thought to be closely involved in binding do indeed appear in or near the binding site for HEPES (shown in Fig. 11.6a), which is on one side of the subunit-subunit interface. The other side of the interface (see Fig. 11.6b) is thought to be formed from the 7 or € subunit for one site, and from the 5 subunit for the other site.
In the AChBP, all the subunits are the same, and for the most part their interface residues do not align in any very convincing way with the y, e, or 8 subunits. The one exception is W53 (see Fig. 11.2a), which aligns with W55 in the mouse y, e, and 8 subunits. The 7 W55 residue contributes to the binding of the Naja naja a-toxin (99), but no binding-gating studies have been done on mutations at this position. An additional problem on this side of the interface is that stated by Brejc et al. (25): "the loop F region has an unusual conformation, but as it is relatively weakly resolved, its precise analysis is difficult."
eD175N. Position 175 in the mouse € subunit was thought to be of interest because of studies by Czajkowski et al. on the homologous position in the 8 subunit, SD180 (100, 101). They mutated all of the aspartate and glutamate residues to asparagine or glu-tamine, respectively, in a region known to be proximal to the binding site from crosslinking studies. The greatest effect seen was an 80fold increase in the EC50 value for ACh produced by the SD180N mutation.
Akk et al. (92) constructed the homologous mouse mutation eD175N to test the effects on the rate constants of binding and gating. By use of high concentrations of ACh and Popen curves, the EC50 value of ¿D175N was found to
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