Genetically-targeted photorelease of endocannabinoids enables optical control of GPR55 in pancreatic β-cells

Abstract

Fatty acid amides (FAAs) are a family of second-messenger lipids that target cannabinoid receptors, and are known mediators of glucose-stimulated insulin secretion from pancreatic β-cells. Due to the diversity observed in FAA structure and pharmacology, coupled with the expression of at least 3 different cannabinoid G protein-coupled receptors in primary and model β-cells, our understanding of their role is limited by our inability to control their actions in time and space. To investigate the mechanisms by which FAAs regulate β-cell excitability, we developed the Optically-Cleavable Targeted (OCT)-ligand approach, which combines the spatial resolution of self-labeling protein (SNAP-) tags with the temporal control of photocaged ligands. By linking a photocaged FAA to an o-benzylguanine (BG) motif, FAA signalling can be directed towards genetically-defined cellular membranes. We designed a probe to release palmitoylethanolamide (PEA), a GPR55 agonist known to stimulate glucose-stimulated insulin secretion (GSIS). When applied to β-cells, OCT-PEA revealed that plasma membrane GPR55 stimulates β-cell Ca²⁺ activity via phospholipase C. Moving forward, the OCT-ligand approach can be translated to other ligands and receptors, and will open up new experimental possibilities in targeted pharmacology.

Fig. 1. OCT-ligands allow genetic targeting of photocaged FAAs. (a) Schematic depiction of the OCT-ligand approach. Photocaged FAAs can be spatially enriched at the site of SNAP-tag expression and then released on demand to activate nearby GPR55 receptors and their downstream effector pathways. (b) Molecular schematic of OCT-PEA tethering and photolysis. OCT-PEA can be tethered to SNAP-tags and uncaged with UV-A (365 nm) irradiation, releasing PEA to activate GPR55. This stimulation increases intracellular Ca²⁺ levels via Gα_q/11 and PLC.

Fig. 2. Synthesis and characterization of OCT-PEA. (a) Chemical synthesis of OCT-PEA. (b) UV-VIS absorbance scan showing a time-course of OCT-PEA (20 μM in DMSO) uncaging with 365 nm LED irradiation. A rightward (bathochromic) shift in the main absorbance peak (λ_max) was observed as illumination generated the uncaged species. (c) Absorbance at 360 nm over time of OCT-PEA (20 μM in DMSO) uncaging with 365 nm LED (black), 405 nm (magenta), 470 nm (blue), and 565 nm (green) LEDs. N = 3 samples for each. Shaded error bars = mean ± SEM.

Fig. 3. INS-1 cells express GPR55 and respond to PEA. (a) Immunofluorescence images of fixed INS-1 cells stained for GPR55 (green) and insulin (red). Nuclei were labelled with DAPI (blue). Scale bar = 10 μm. (b) Fluorescence intensity profile plotted across the yellow arrow shown in panel A. (c) Fluorescent Ca²⁺ imaging using R-GECO showed that PEA addition (5 μM) increased [Ca²⁺]_i. Displayed as Ca²⁺ traces from five representative cells. (d) Average [Ca²⁺]_i traces for PEA addition under standard conditions (black, 5 μM, N = 612, T = 10), overlaid with average [Ca²⁺]_i traces for PEA addition following pre-incubation with a GPR55 antagonist (CID16020046, 5 μM, blue, N = 449, T = 6) or PLC inhibitor (U73122, 5 μM, red, N = 296, T = 4), which blocked PEA's effect. UV-A irradiation (375 nm) shown as purple bars, and KCl (25 mM) was applied at the end of each experiment. (e) Bar graph displaying the fold change in Ca²⁺ oscillation frequency induced by compound stimulation. Vehicle addition (DMSO, 0.1% v/v, grey, N = 418, T = 6) did not stimulate [Ca²⁺]_i. The inactive PLC inhibitor analogue (U73343, 5 μM, green, N = 511, T = 7) did not block the effect of PEA. Error bars = mean ± SEM. **P < 0.01, *P < 0.05, ns = P > 0.05, P values reported in Table S2.

Fig. 4. Targeted uncaging of OCT-PEA on the INS-1 cell surface. (a) Pre-incubation with DMSO (0.1% v/v, 2 h) maintains surface SNAP-tag labelling (A488, green, left), whereas pre-incubation with OCT-PEA (5 μM, 2 h) abolished dye labelling (right). Nuclei stained with Hoechst-33342 (blue). Scale bar = 10 μm. (b) Fluorescent Ca²⁺ imaging using R-GECO showed that OCT-PEA (5 μM, 2 h, black, N = 352, T = 8) increased the average [Ca²⁺]_i in INS-1 cells. Overlaid with averages in the presence of a GPR55 antagonist (CID16020046, 5 μM, blue, N = 212, T = 4) or PLC inhibitor (U73122, 5 μM, red, N = 173, T = 4), which reduced the effect of OCT-PEA. UV-A irradiation (375 nm) shown as purple bars, and KCl (25 mM) was applied at the end of each experiment. (c) Heat map showing individual Ca²⁺ traces from fifty representative cells which were pre-incubated with OCT-PEA (5 μM, 2 h). Cells normalized to the KCl response. (d) Comparison bar graph of fold change in oscillation frequency in response to OCT-PEA uncaging across different conditions. Uncaging in the presence of GPR55 antagonist (CID16020046, 5 μM, purple, N = 212, T = 4), PLC inhibitor (U73122, 5 μM, blue, N = 173, T = 4), SNAP-Surface® Block (20 μM, yellow, N = 278, T = 5), SNAP-Cell® Block (10 μM, orange, N = 248, T = 4), pDisplay™-SNAP^C145A (red, N = 244, T = 5) or pDisplay™-HALO (magenta, N = 330, T = 4) reduced the probe's effect on oscillation frequency. The inactive PLC inhibitor analogue (U73343, 5 μM, green, N = 357, T = 6) did not block the effect of OCT-PEA. Vehicle treatment (DMSO, 0.1% v/v, grey, N = 247, T = 4) did not change the oscillation frequency in response to UV-A irradiation. (e and f) Uncaging OCT-PEA in INS-1 cells transfected with pDisplay™-SNAP^C145A reduced the effect of the probe, displayed as (e) average trace of all cells and (f) heat map of representative traces from fifty cells, normalized to the KCl response. (g–i) Targeted uncaging on half of the field of view, displayed as (g) uncaging ROI, (h) bar graph comparing fold change in oscillation frequency between irradiated (N = 145, T = 5) and nonirradiated regions (N = 132, T = 5), and (i) heat map of representative traces from one trial, normalized to KCl response. Error bars = mean ± SEM. *P < 0.05, **P < 0.01, ns = P > 0.05, P values reported in Table S3.