Figure 1.

Imaging binding and unbinding of dermorphin A594. A, FLAG-MOR in HEK 293 cells were labeled with M1 anti-FLAG antibody-conjugated Alexa-488 (M1 A488) to visualize receptors localized on the plasma membrane. Dermorphin Alexa-594 (100 nm, derm A594) was applied for 90 s (wash in at t = −90 washout at t = 0 in A). derm A594 binding and unbinding were assessed by imaging M1 A488 and derm A594 every 2.5 s with images from selected time points shown. Scale bar, 20 μm. B, Ratiometric imaging of derm A594: M1 A488 (R/G) intensity was used to quantify binding and unbinding of agonist to receptor from the experiment shown in A. C, Specificity of binding was assessed by determining the relative fluorescence intensity (R/G) of derm A594 bound immediately after application of derm A594 (5 min, 500 nm) under control conditions or on cells pretreated with β-CNA (1 μm, 5 min) and bathed in naloxone (10 μm). Data are mean ± SEM (ctrl, n = 5; β-CNA/naloxone, n = 6).