Research ArticleGPCR SIGNALING

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

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Sci. Signal.  03 Nov 2015:
Vol. 8, Issue 401, pp. ra110
DOI: 10.1126/scisignal.aac7419

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Agonist control of GPCR voltage sensitivity

Most G protein–coupled receptors (GPCRs) are activated by ligand binding, but some are also affected by changes in plasma membrane potential, which can either enhance or inhibit GPCR-mediated signaling. Through FRET-based experiments in single cells, Rinne et al. found that depolarization enhanced signaling by the M3 muscarinic acetylcholine receptor when the receptor was bound to the agonists choline or pilocarpine; however, depolarization attenuated M3 receptor signaling when either carbachol or acetylcholine was bound. Molecular docking simulations showed that each group of agonists adopted a distinct binding position. Mutation of a critical residue in the binding pocket changed the binding position of carbachol and switched the response of the carbachol-bound receptor so that signaling was enhanced by membrane depolarization. Together, these data suggest that the binding mode of the agonist determines whether membrane potential changes will enhance or attenuate GPCR signals. These results provide a potential molecular mechanism for drugs that are agonists of specific GPCRs, yet have distinct effects.


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.

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