The mode of agonist binding to a G protein-coupled receptor switches the effect that voltage changes have on signaling.

Signaling by many heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) is either enhanced or attenuated by changes in plasma membrane potential. To identify structural correlates of the voltage sensitivity of GPCR signaling, we chose muscarinic acetylcholine receptors (the M1, M3, and M5 isoforms) as a model system. We combined molecular docking analysis with Förster resonance energy transfer (FRET)-based assays that monitored receptor activity under voltage clamp conditions. When human embryonic kidney (HEK) 293 cells expressing the individual receptors were stimulated with the agonist carbachol, membrane depolarization enhanced signaling by the M1 receptor but attenuated signaling by the M3 and M5 receptors. Furthermore, whether membrane depolarization enhanced or inhibited receptor signaling depended on the type of agonist. Membrane depolarization attenuated M3 receptor signaling when the receptor was bound to carbachol or acetylcholine, whereas depolarization enhanced signaling when the receptor was bound to either choline or pilocarpine. Docking calculations predicted that there were two distinct binding modes for these ligands, which were associated with the effect of depolarization on receptor function. From these calculations, we identified a residue in the M3 receptor that, when mutated, would alter the binding mode of carbachol to resemble that of pilocarpine in silico. Introduction of this mutated M3 receptor into cells confirmed that the membrane depolarization enhanced, rather than attenuated, signaling by the carbachol-bound receptor. Together, these data suggest that the directionality of the voltage sensitivity of GPCR signaling is defined by the specific binding mode of each ligand to the receptor.