Abstract
G protein-coupled receptors (GPCRs) are proteins that span the cell membrane seven times. They are stimulated by extracellular agonists and activate heterotrimeric G proteins to elicit intracellular responses. Recent evidence suggests that GPCRs are voltage-sensitive: They exhibit gating currents similar to ion channels and respond to depolarization of the plasma membrane with changes in agonist affinity. We used a FRET-based biosensor of the α2A adrenergic receptor to analyze voltage dependence of receptor activation in HEK 293 cells by means of voltage-clamp recording. The biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor activation was measured as decrease in FRET ratio. Receptor stimulation by NE was reduced by membrane depolarization and enhanced by hyperpolarization. This effect was present in wilde-type receptors and transduced to the level of G protein activation, which we determined in a FRET assay that directly detects Gαi protein activation. Depolarization-mediated inhibition of NE activated receptors was strong at low concentrations (500 nM: 60 % inhibition) but almost undetectable at saturating agonist concentrations (100 μM: 9 % inhibition). Both agonist-induced and hyperpolarization-induced receptor activation exhibited a similar monoexponential time course. For both activation modes, speed of activation was primarily dependent on agonist concentration, indicating that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 μM, VM=-90 mV) resulted in a FRET response that was inhibited by 40 % at +60 mV. In contrast to NE, strong receptor-inhibition at +60 mV was present even at super-saturating concentrations of clonidine (100 μM). Therefore we conclude that negative membrane potentials promote active conformations of the α2A adrenergic receptor, increase affinity of full agonists and enhance G protein signaling.
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