The human muscarinic acetylcholine receptors are a member of the major subfamily of G-protein-coupled receptors. The M1 subtype (M1R) is selectively distributed in the forebrain and has been linked to higher cognitive function, such as memory formation and spatial navigation. M1R has recently emerged as a promising therapeutic target in Alzheimer’s disease and schizophrenia. Several lines of evidence suggest that the dynamic reorganization of M1R in the membrane, not its endocytic trafficking, control its spatiotemporal signaling in response to stimulation. To investigate this dynamic response at the level of individual molecular species, human embryonic kidney (HEK)-293 cells were stably transfected with hemagglutinin (HA)-tagged M1Rs in order to overexpress the receptor and enable QDot detection. The HA epitope (YPYDVPDYA), fused to the extracellular N-terminus of M1R, was labeled with biotinylated anti-HA antibody fragments and streptavidin-conjugated Qdots. Single Qdot-tagged M1R proteins were tracked at the basolateral membrane at acquisition speeds of 10-150 Hz over a 1-minute period. A combination of ImageJ TrackMate plugin and custom MATLAB routines were used to extract and analyze single-molecules trajectories of Qdot-M1Rs. Initial analysis revealed that at basal conditions M1Rs diffuse laterally at an average rate of 0.031 µm<sup>2</sup>/s. In contrast, acute acetylcholine stimulation induced significant diffusional slowing of M1Rsby nearly 50%. Additionally, multiple pools of transporters were detected based on transient diffusivity under both conditions. Current experiments are aimed at investigating transient M1R interactions with the juxtamembrane actin cytoskeleton corrals under both basal and activated conditions through single Qdot tracking and superresolution STORM imaging. We hope that these experiments will provide valuable single-molecule mechanistic insights into the dynamic response of M1R to activation. Ultimately, the elucidation of nanoscale organizational complexity of M1R may lead to a paradigm shift in current drug development strategies that benefits the end patient.

Elucidating the membrane diffusion dynamics of muscarinic-1 acetylcholine receptor (M1R) with quantum dots

256th National Meeting (2018)

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