Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells − The Impact of Ligands and Membrane Disruptors

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different liganded states together with spectroscopic studies and molecular dynamics simulations have revealed a rich conformational landscape of GPCRs. However, their supramolecular spatio-temporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M ₁ receptor (M ₁ R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M ₁ R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M ₁ receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 μm ² /s), slow (∼0.04 μm ² /s), and fast (∼0.14 μm ² /s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6-ceramide led to significant changes for CD86 while leaving the diffusion of M ₁ R unchanged, indicating that M ₁ receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression, and the binding of orthosteric ligands; in particular the agonist carbachol elicited a large increase in the fraction of M ₁ R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.