A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (DeltaF/F) approximate to10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for DeltaF agreed well with those for epibatidine-induced currents, but were shifted approximate to100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alphagamma-binding site than to the alphadelta site and (it) ACh binds with reverse-site selectivity, these data suggest that DeltaF monitors an event linked to binding specifically at the alphadelta-subunit interface. In experiments with flash-applied agonists, the earliest detectable DeltaF occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of DeltaF occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from DeltaF is complete before the slowest phase of recovery from desensitization (time constant approximate to250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alphadelta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.