| dc.description.abstract | 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 human muscarinic-1 acetylcholine receptors (hM1AchR) in order to overexpress the receptor and enable quantum-dot (QDot) detection. The HA epitope (YPYDVPDYA), fused to the extracellular N-terminus of hM1AchR, was labeled with biotinylated anti-HA antibody fragments and streptavidin-conjugated Qdots. Single Qdot-tagged hM1AchR 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-molecule trajectories of Qdot-hM1AchRs. Initial analysis revealed that at basal conditions, hM1AchRs diffuse laterally at an average rate of 0.031 µm2/s. In contrast, acute acetylcholine stimulation induced diffusional slowing of hM1AchRs by nearly 40%. Additionally, multiple pools of transporters were detected based on transient diffusivity under stimulated conditions. Investigation of transient hM1AchR interactions with the cellular membrane under both basal and activated conditions through single Qdot tracking and total internal reflection fluorescence (TIRF) imaging was completed. These experiments provided valuable single-molecule mechanistic insights into the dynamic response of hM1AchR to activation. Ultimately, the elucidation of nanoscale organizational complexity of hM1AchR may lead to a paradigm shift in current drug development strategies that benefits the end patient. | |
