The five muscarinic acetylcholine receptors (mAChRs M1-M5) are an important family of class A G protein-coupled receptors (GPCRs) that are widely expressed throughout the central and peripheral nervous systems. Upon activation by the endogenous ligand acetylcholine, mAChRs recruit transducer proteins to transmit an extracellular signal into the cell. The five mAChRs display a different preference for transducer proteins as the M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins whereas the M2 and M4 mAChRs preferentially couple to Gi/o proteins1. Despite this, there is evidence of pleiotropic G protein coupling at the mAChRs, particularly at the M2 mAChR2. These previous experiments have relied on the use of recombinant cell lines and second messenger analysis where individual G proteins are removed through the use of a range of techniques such as pertussis toxin, siRNA, and genetic knockout. Furthermore, other experiments have suggested that different oligomeric states of the M2 mAChR could be responsible for the differential signaling. Given the multitude of factors that drive second messenger responses, understanding whether a single receptor can functionally interact with different G proteins remains to be comprehensively determined. Additionally, whether a preference for different Gi isoforms coupling to the M2 mAChR exists remains unexplored. To that end, we have reconstituted the M2 mAChR into high density lipoproteins (rHDLs) otherwise known as nanodiscs, a reductionist approach well suited for this line of enquiry. Through the use of purified wild type heterotrimeric G proteins and M2 mAChR rHDLs we have begun to investigate the role of different G protein subtypes in coupling to the M2 mAChR and their ability to stabilize the active state in the presence of different orthosteric ligands.