Schematic portrayal of the proposed arrangement of IP₃Rs underlying the generation of blips and puffs. (A) Monochrome image of resting fluo-4 fluorescence in an SH-SY5Y cell observed using TIRF microscopy, mapping sites that displayed single-channel blip activity in response to photoreleased IP₃ (black filled circles) and sites that displayed puffs (white circles). (B) The trace shows experimental data of activity recorded from a blip site. Cartoons illustrate our suggested arrangement and activity of IP₃R channels corresponding to each of the three blips during the trace. Channels open during each blip are shown in red, and closed channels are shown in black. Both are sensitized to IP₃ by binding to an as yet unidentified anchoring site (depicted in green). Motile, functionally unresponsive IP₃Rs are shown in gray. (C) Corresponding trace and cartoons illustrate the localization and activity of IP₃R channels during puffs. Gray lines indicate fluorescence levels corresponding to integer multiples of the single-channel (blip) level. The first puff (left) involves simultaneous opening of six channels at its peak, and the second puff (right) only two channels. The channels contributing to puffs are believed to be distributed across clusters with a diameter of roughly 400 nm.