Quantal Ca(2+) release mediated by very few IP(3) receptors that rapidly inactivate allows graded responses to IP(3).

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that link extracellular stimuli to Ca2+ signals. Ca2+ release from intracellular stores is "quantal": low IP3 concentrations rapidly release a fraction of the stores. Ca2+ release then slows or terminates without compromising responses to further IP3 additions. The mechanisms are unresolved. Here, we synthesize a high-affinity partial agonist of IP3Rs and use it to demonstrate that quantal responses do not require heterogenous Ca2+ stores. IP3Rs respond incrementally to IP3 and close after the initial response to low IP3 concentrations. Comparing functional responses with IP3 binding shows that only a tiny fraction of a cell's IP3Rs mediate incremental Ca2+ release; inactivation does not therefore affect most IP3Rs. We conclude, and test by simulations, that Ca2+ signals evoked by IP3 pulses arise from rapid activation and then inactivation of very few IP3Rs. This allows IP3Rs to behave as increment detectors mediating graded Ca2+ release.

Rossi AM ，Riley AM ，Dupont G ，Rahman T ，Potter BVL ，Taylor CW ... -  《Cell Reports》

Dissociation of inositol 1,4,5-trisphosphate from IP(3) receptors contributes to termination of Ca(2+) puffs.

Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.

Smith HA ，Taylor CW 《-》

Comparison of Ca(2+) puffs evoked by extracellular agonists and photoreleased IP(3).

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.

Lock JT ，Smith IF ，Parker I 《-》

IP(3) receptors: An "elementary" journey from structure to signals.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are large tetrameric channels which sit mostly in the membrane of the endoplasmic reticulum (ER) and mediate Ca2+ release from intracellular stores in response to extracellular stimuli in almost all cells. Dual regulation of IP3Rs by IP3 and Ca2+ itself, upstream "licensing", and the arrangement of IP3Rs into small clusters in the ER membrane, allow IP3Rs to generate spatially and temporally diverse Ca2+ signals. The characteristic biphasic regulation of IP3Rs by cytosolic Ca2+ concentration underpins regenerative Ca2+ signals by Ca2+-induced Ca2+-release, while also preventing uncontrolled explosive Ca2+ release. In this way, cells can harness a simple ion such as Ca2+ as a near-universal intracellular messenger to regulate diverse cellular functions, including those with conflicting outcomes such as cell survival and cell death. High-resolution structures of the IP3R bound to IP3 and Ca2+ in different combinations have together started to unravel the workings of this giant channel. Here we discuss, in the context of recently published structures, how the tight regulation of IP3Rs and their cellular geography lead to generation of "elementary" local Ca2+ signals known as Ca2+ "puffs", which form the fundamental bottleneck through which all IP3-mediated cytosolic Ca2+ signals must first pass.

Smith HA ，Thillaiappan NB ，Rossi AM 《-》

IP(3) receptors - lessons from analyses ex cellula.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment ('ex cellula'), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells have demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from this store. Radioligand binding confirmed that the 4,5-phosphates of IP3 are essential for activating IP3Rs, and facilitated IP3R purification and cloning, which paved the way for structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope have established that IP3Rs have a large conductance and select weakly between Ca2+ and other cations. Structural analyses are now revealing how IP3 binding to the N-terminus of the tetrameric IP3R opens the pore ∼7 nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved domains contributed by structures associated with the pore and cytosolic domains, which together contribute to a Ca2+-binding site. These structural analyses provide evidence to support the suggestion that IP3 gates IP3Rs by first stimulating Ca2+ binding, which leads to pore opening and Ca2+ release.

Rossi AM ，Taylor CW 《-》