Real-time detection of ?_1A-AR movement stimulated by phenylephrine in single living cells

Aim: To investigate the movement of alpha(1A)-adrenergic receptors(alpha(1A)-AR) stimulated by agonist, phenylephrine (PE), and the dynamics of receptor movement in real time in single living cells with millisecond resolution. Methods: We labeled alpha(1A)-AR using the monoclonal, anti-FLAG (a kind of tag) antibody and Cy3-conju-gated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. Results: The specific detection of alpha(1A)-AR on the surface of living HEK293A-alpha(1A) cells was achieved. alpha(1A)-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20 min, apparent colocalization was found between alpha(1A)-AR and F-actins. After 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A-alpha(1A) cells were recorded, and their velocity was calculated. Conclusion: The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect alpha(1A)-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.