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High spatio‐temporal resolution measurement of A1R and A2AR interactions combined with Iem‐spFRET and E‐FRET methods

A1R‐A2AR heterodimers regulate striatal glutamatergic neurotransmission. However, few researches about kinetics have been reported. Here, we combined Iem‐spFRET and E‐FRET to investigate the kinetics of A1R and A2AR interaction. Iem‐spFRET obtains the energy transfer efficiency of the whole cell. E‐FRET gets energy transfer efficiency with high spatial resolution, whereas, it was prone to biases because background was easily selected due to manual operation. To study the interaction with high spatio‐temporal resolution, Iem‐spFRET was used to correct the deviation of E‐FRET. In this paper, A1R and A2AR interaction was monitored, and the changes of FRET efficiency of the whole or/and partial cell membrane were described. The results showed that activation of A1R or A2AR leads to rapid aggregation, inhibition of A1R or A2AR leads to slow segregation, and the interaction is reversible. These results demonstrated that combination of Iem‐spFRET and E‐FRET could measure A1R and A2AR interaction with high spatio‐temporal resolution.