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. Author manuscript; available in PMC: 2020 Nov 3.
Published in final edited form as: J Am Chem Soc. 2019 Oct 10;141(42):16544–16547. doi: 10.1021/jacs.9b05978

Figure 2.

Figure 2.

(A) Representative traces of fluorescence increase F/F0 of live MIN6 cells pretreated with the cell permeant calcium dye Fluo4/AM (5 μM) in the presence of cg2-AG ([cg2-AG] = 10 μM, n = 6, cyan trace) and subjected to flash photolysis (λex = 488 nm, λuncaging = 375 nm) with and without the CB1 antagonist rimonabant (1 μM; [cg2-AG] = 10 μM, n = 6, pink trace). (B) Bar graphs summarizing fluorescence intensity increase F/F0 of the calcium dye Fluo4/AM (5 μM) in MIN6, treated with cg2-AG and subjected to flash photolysis in the absence ([cg2-AG] = 10 μM, n = 136) or presence of rimonabant (1 μM; [cg2-AG] = 10 μM, n = 79). The CB1 agonist WIN55 was used as a positive control ([WIN55] = 10 μM, n = 84). (C) Calcium imaging of MIN6 cells upon the uncaging of cg2-AG (10 μM) ± preincubation with rimonabant (1 μM). (D) Photomicrograph showing a recording pipet patched onto a MIN6 cell dispersed on a coverslip. (E) Currents generated in a whole-cell recording of a MIN6 cell elicited by holding the cell at −60 mV and giving a series of 1 s prepulses ranging from −50 mV to −140 mV (in 10 mV increments) and stepping back to −60 mV before, after photostimulation (0.6 mW for 1 min, and during perfusion with 300 μM diazoxide). (F) Representative traces of photoinduced currents by released 2-AG, KATP opener diazoxide, or GIRK channel blocker tertiapin (upper trace). After a seal was formed and the whole-cell configuration was obtained, cells were perfused with cg2-AG (10 μM) for 10 min before activating with a 375 nm light pulse. After the current returned to resting level, the cells were perfused with diazoxide, which generated a robust outward current. Vhold = −60 mV (lower trace). The outward current induced by the uncaging of cg2-AG was blocked by pretreatment with GIRK channel blocker tertiapin (100 nM) for 15 min. (G) I/V curves generated in MIN6 cells in the presence of cg2-AG (10 μM, ± UV light) or diazoxide (300 μM). (H) Bar graphs summarizing the effects of cg2-AG in absence or presence of tertiapin. Data points represent the mean ± SEM. Cell numbers are indicated. Unpaired t test, t = 2.551, P = 0.0447.