Real-Time Optical Control of CB1 Receptor Signaling <i>In Vitro</i> with Tethered Photoswitchable (−)-<i>trans</i>-Δ⁹-Tetrahydrocannabinol Derivatives

Understanding the intricacies of the endocannabinoid system is hindered by the lack of tools to target specific pools of CB1 receptors (CB1Rs) across diverse neural circuits associated with mood, motor function, cognition, and other physiological processes. Herein, we introduce the first photoswitchable, orthogonal remotely tethered cannabinoid ligand, PORTL-THC₂₄, designed to achieve cell-specific and reversible control of CB1R signaling with high spatial and temporal resolution, thereby overcoming the limitations of conventional freely diffusible ligands. PORTL-THC₂₄ was selectively tethered to membrane-anchored SNAP-tags expressed in live cells, and provided reversible optical control of CB1R signaling when photoswitched by UV-A irradiation. We validated the functionality of PORTL-THC₂₄ in live Neuro2a cells using a novel real-time cAMP imaging assay, demonstrating light-dependent and reversible modulation of endogenously expressed CB1R activity. Additionally, we demonstrated that SNAP-tethered PORTL-THC₂₄ does not induce CB1R internalization, distinguishing it from conventional, freely diffusible agonists. Our results establish PORTL-THC₂₄ as a powerful tool for optical control of CB1R in a spatially restricted manner, setting the stage for dissecting CB1R function in complex settings and advancing the study of cannabinoid signaling across various physiological and pathological contexts.