Diffusion and Oligomerization States of the Muscarinic M 1 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 ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal 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 the diffusion of M 1 R remained 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.