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

Abstract

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.

Membrane potentials regulating GPCRs: insights from experiments and molecular dynamics simulations

“…In recent years, V m has been experimentally demonstrated to affect the conformation, function and transmitted signals of a range of GPCRs [26, 23, 19, 27, 18••]. Voltage-related effects have, for instance, been reported for the muscarinic, adrenergic, and purinergic receptor families [20, 18••, 19].…”

“…Voltage-related effects have, for instance, been reported for the muscarinic, adrenergic, and purinergic receptor families [20, 18••, 19]. In most of the earlier work, evidence for voltage regulation was obtained indirectly, and measurements often relied on ionic current through downstream G-protein coupled inward rectifying potassium channels (GIRK) [28] or the use of intracellular calcium-sensitive dyes [26].…”

“…These channel proteins contain specialised voltage-sensing domains, which are capable of inducing large-scale conformational transitions that gate the channels open or closed, even under small changes of V m [17]. By contrast, voltage-related effects on other membrane proteins such as GPCRs seem less intuitive, although a number of studies have reported compelling evidence for a broad range of V m -induced phenomena in GPCRs [11, 18••, 19, 20, 21, 22•] (for review see Ref [23]). Many important class A GPCR drug targets are expressed in excitable tissue, for instance the aminergic, opioid, adrenergic and purinergic receptors.…”

Pharmacological Conversion of a Cardiac Inward Rectifier into an Outward Rectifier Potassium Channel

“…Therefore, we postulated that variations in membrane potential directly induce conformational changes in the ligand-binding site that facilitate the binding of some agonists and hinder the binding of other agonists . This proposal is supported by findings in D2 dopaminergic (Sahlholm et al, 2008(Sahlholm et al, , 2011, a2A adrenergic (Rinne et al, 2013), and Gq-coupled muscarinic receptors, M 1 muscarinic receptor, M 3 muscarinic receptor, and M 5 muscarinic receptor (Rinne et al, 2015). Agonist-specific voltage sensitivity was termed to denote the relationship between specific agonists and voltage (Sahlholm et al, 2008(Sahlholm et al, , 2011Navarro-Polanco et al, 2011;MorenoGalindo et al, 2016).…”

“…Gating currents result from the movement of gating charges across the membrane electrical field and represent voltagedependent conformational changes in the receptor in response to changes in membrane potential, representing the most important evidence about the voltage sensitivity of these muscarinic receptors. Likewise, other GPCRs have been noted to display intrinsic voltage dependence (Dekel et al, 2012;Rinne et al, 2013Rinne et al, , 2015.…”