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 β19′ 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 (ΔF/F) ≈10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose–response relations for ΔF agreed well with those for epibatidine-induced currents, but were shifted ≈100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the αγ-binding site than to the αδ site and (ii) ACh binds with reverse-site selectivity, these data suggest that ΔF monitors an event linked to binding specifically at the αδ-subunit interface. In experiments with flash-applied agonists, the earliest detectable ΔF occurs within milliseconds, i.e., during activation. At low [ACh] (≤ 10 μM), a phase of ΔF occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from ΔF is complete before the slowest phase of recovery from desensitization (time constant ≈250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the αδ-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.