UC Irvine

The inositol trisphosphate (IP₃) signaling pathway evokes local Ca²⁺ signals (Ca²⁺ puffs) that arise from the concerted openings of clustered IP₃ receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP₃ into the cytosol. Photorelease of IP₃ from a caged precursor provides a convenient and widely employed means to study the final stage of IP₃-mediated Ca²⁺ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP₃. Here, we address whether Ca²⁺ puffs evoked by photoreleased IP₃ fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP₃ analog, i-IP₃. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1μm), suggesting that IP₃ from both sources accesses the same, or closely co-localized clusters of IP₃Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP₃ uniformly throughout the cytoplasm, our results imply that IP₃ generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP₃Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.