Real-time detection of alpha1A-AR movement stimulated by phenylephrine in single living cells.
OBJECTIVE
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-conjugated 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.


CONCLUSIONS
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.
Xu
Ning
N
Institute of Vascular Medicine, Peking University Third Hospital, and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100083, China.
Liang
Zhang-Yi
ZY
Xu
Ming
M
Guan
Ying-Hua
YH
He
Qi-Hua
QH
Han
Qi-De
QD
Zhao
Xin-Sheng
XS
Zhang
You-Yi
YY
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line / Q000187:drug effects*; D006801:Humans; D051379:Mice; D010656:Phenylephrine / Q000494:pharmacology*; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D018340:Receptors, Adrenergic, alpha-1; D010656:Phenylephrine