Single-Molecule Analysis of the Supramolecular Organization of the M₂ Muscarinic Receptor and the Gα_i1 Protein

G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M₂ muscarinic receptor (M₂R) and G_i1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1–4 copies of fused eGFP. The photobleaching patterns of eGFP-M₂R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-G_i1 were indicative of a hexamer and unaffected by GTPγS. A complex of M₂R and G_i1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of G_i1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gα_i1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gα_i1 was predominantly dimeric. The oligomeric nature of G_i1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gα_i1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M₂R and holo-G_i1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of G_i1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations