Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3

Abstract The inositol trisphosphate (IP 3 ) signaling pathway evokes local Ca 2+ signals (Ca 2+ puffs) that arise from the concerted openings of clustered IP 3 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 IP 3 into the cytosol. Photorelease of IP 3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP 3 -mediated Ca 2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP 3 . Here, we address whether Ca 2+ puffs evoked by photoreleased IP 3 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 IP 3 analog, i-IP 3 . The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1 μm), suggesting that IP 3 from both sources accesses the same, or closely co-localized clusters of IP 3 Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP 3 uniformly throughout the cytoplasm, our results imply that IP 3 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 IP 3 Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.