Fig. 2.

Calcium influx through TRPV1 blocks MOR1 phosphorylation and internalization. (A and B) Western blots of HEK293 cells coexpressing TRPV1 and MOR1. (A) EGTA (5 mM) chelation of extracellular calcium blocks capsaicin (1 µM) inhibition of MOR1 phosphorylation. (B) Decreasing extracellular calcium from 10 mM to 1 µM rescues MOR1 phosphorylation even with 5 min of TRPV1 activation by capsaicin (1 µM). (C and D) Immunofluorescence of transiently transfected HEK293 cells. (C) Staining of HA-MOR1 in cells coexpressing HA-MOR1-WT and myc-TRPV1 treated with combinations of EGTA (5 mM) for 5 min, capsaicin (1 µM) for 5 min, and DAMGO (1 µM) for 15 min, as indicated. (D) Line plot profile of green intensity (MOR1) along a white line drawn to transect both membranes of cell from associated treatment groups in C. Green, HA-MOR1; blue, DAPI. (Scale bars: 10 µm.)