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IP3, still on the move but now in the slow lane

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Science Signaling  08 Nov 2016:
Vol. 9, Issue 453, pp. fs17
DOI: 10.1126/scisignal.aal1929


In this issue of Science Signaling, Dickinson et al. show that the intracellular messenger inositol 1,4,5-trisphosphate (IP3), which triggers the release of calcium (Ca2+) from the endoplasmic reticulum, is a slowly diffusing local signal, rather than a rapidly diffusing global one. These findings have implications for the understanding of the mechanisms of Ca2+ wave propagation, especially long-range, cell-to-cell propagating Ca2+ waves.

Inositol 1,4,5-trisphosphate (IP3) was discovered in 1983 as a major second messenger that triggers the release of calcium ions (Ca2+) from stores located in the endoplasmic and sarcoplasmic reticulum (ER/SR) (1). IP3 is generated in response to a first (extracellular) messenger that binds to a G protein–coupled receptor or a receptor tyrosine kinase and subsequently activates phospholipase C (PLCγ or PLCβ, respectively). PLC cleaves phosphatidylinositol 4,5-bisphosphate, a plasma membrane–located phosphoinositide, into IP3 that diffuses in the cytoplasm and diacylglycerol (DAG) that remains associated with the plasma membrane and activates protein kinase C. IP3 binds to IP3 receptors (IP3Rs) on the ER/SR, resulting in the opening of these Ca2+-permeable channels and increasing the cytoplasmic Ca2+ concentration ([Ca2+]i). Compared with the action range (~5 μm) and diffusion constant (~38 μm2/s) of Ca2+ in the cytoplasm (2), IP3 has been considered a global cellular messenger with a diffusion constant of ~280 μm2/s (2). Consequently, the diffusion of IP3, rather than the diffusion of Ca2+ itself (3, 4), has been considered the mediator of long-distance signaling in large-diameter cells (mammalian oocytes, ~100 μm; Xenopus oocytes, ~1000 μm) or in the context of cell-to-cell propagating [Ca2+]i changes as intercellular Ca2+ waves.

In this issue, Dickinson et al. (5) provide convincing evidence that IP3 diffusion is much slower than initially thought. They used ultraviolet photoactivation of caged (inactive) and poorly metabolizable IP3 to produce step-like changes in IP3 concentration in SH-SY5Y neuroblastoma cells and investigated the delay in the appearance of “Ca2+ puffs” at various distances away from the photoactivation spot with Ca2+ imaging. Ca2+ puffs are local, elementary Ca2+ release events that result from the activation of a cluster of a few of the total IP3Rs present in a cell (6). Ca2+ puffs are stochastic, noisy events; thus, computational modeling was necessary to fit and describe the data accurately. Strikingly, their results indicate that the diffusion constant of IP3 is less than 10 μm2/s, almost 30 times less than previously reported.

IP3 has few known binding sites other than those on IP3Rs, which are present throughout the ER in immobile clusters. Yet, only a small fraction are activated to produce Ca2+ puffs (7). From these observations, the authors speculate that there are two populations of IP3Rs, (i) a small clustered pool that mediates elementary Ca2+ signals like puffs and blips (blips are mediated by a single IP3R) and (ii) a larger pool that buffers IP3 and slows its diffusion. Furthermore, they speculate that the IP3Rs that assist in buffering cytoplasmic IP3 are functionally silent receptors because they may not be bound to four IP3 molecules, which is required for the channel to open (8). Thus, partly occupied IP3Rs may buffer cytoplasmic IP3. As expected, increasing the concentration of photoreleased IP3 produced a (slight) increase of the IP3 diffusion constant because free IP3 becomes more prevalent, leading to less-hindered, faster diffusion [as was also reported for Ca2+ by Allbritton et al. (2)].

The three isoforms of IP3Rs have distinct IP3 affinities. Although the affinity differences are not large (9), different IP3R isoforms may have some effect on IP3 diffusion, and this aspect deserves a closer look. Also, the concentration of IP3Rs is a factor that will determine the buffering capacity: Cells with low IP3R density, such as cardiomyocytes, will display faster, less-hindered IP3 diffusion, and cells with high IP3R density, such as Purkinje cells, will display a slower IP3 diffusion.

A question that remains open from the Dickinson et al. study concerns the IP3 concentrations attained upon photoactivation. Calibrating the amount of photoreleased IP3 is difficult and was not performed. IP3 triggers Ca2+ release in a concentration range that varies between 30 nM and 10 μM with a half-maximal response at 240 nM (10). Photorelease of larger IP3 amounts than achieved by Dickinson et al. or bolus injections of IP3, such as those used to trigger intercellular Ca2+ waves, may result in IP3 diffusion that is faster than 10 µm2/s. All computational modeling of intercellular Ca2+ waves has been based on 280 μm2/s as a diffusion constant for IP3. Do we need to completely rethink our interpretations of studies that based their conclusions on too fast a diffusion of IP3? For small cells or localized, microdomain IP3-mediated Ca2+ signaling, as occurs in neuronal dendrites, it is likely that nothing needs to be reanalyzed because the size of these cells and subcellular structures inherently limit the range of IP3 action. In larger cells, like oocytes, IP3-mediated Ca2+ signaling forms the basis of Ca2+ waves and oscillations that occur during fertilization. Here, as well as in other cell types, the models have assumed that IP3, because of its fast diffusion from its point of generation, first primes the IP3Rs to make them more sensitive to Ca2+, which arrives with some delay (11). IP3Rs are generally enriched in the periphery of the oocyte, and their distribution changes during oocyte maturation (12). This would be expected to create spatial heterogeneity in IP3 diffusion speed, the effect of which needs to be verified in experimental work and revised computational modeling studies.

Intercellular Ca2+ waves are long-range, cell-to-cell [Ca2+]i changes that propagate with a speed in the order of 10 to 20 μm/s (4). This communication relies on two pathways: One is mediated by gap junction channels that directly connect the cytoplasm of adjacent cells, and the other pathway involves the release of an extracellular messenger and paracrine signaling to the next cell (Fig. 1). The gap junction–based pathway involves the diffusion of either Ca2+ or IP3 or both in the cytoplasm and the subsequent passage of these messengers through the junctional channel. Both IP3 and Ca2+ can permeate through gap junctions, which are assemblies of large pore channels (10 to 20 Å) composed of connexin subunits. It has been assumed, on the basis of Allbritton et al. (2), that IP3 diffuses faster than Ca2+ and therefore reaches the gap junction first (4). Examples in which Ca2+ was pinpointed as the gap junction–permeating messenger exist but are sparse, supporting the view of IP3 as the major long-range messenger. This view needs to be reconsidered. Because there are two pathways for intercellular Ca2+ wave propagation, the slower diffusion of IP3 may mean that the paracrine pathway, involving extracellular signals like adenosine 5′-triphosphate (ATP), plays a larger role than previously thought. The intra- and extracellular signaling pathways are strongly intertwined: (i) They both use IP3 as an intermediate signal; (ii) connexins play a role in junctional IP3 diffusion but also in ATP release; and (iii) IP3 and extracellular ATP can be regenerated in a Ca2+-dependent manner (4). Hence, additional modeling will be necessary to determine and understand the effect of 30-fold slower IP3 diffusion. In any case, we have to give up our view of IP3 as the hare and Ca2+ as the turtle.

Fig. 1

Who is first at the gap junction: IP3 or Ca2+? Mechanistic considerations of intercellular Ca2+ wave propagation have always assumed that IP3 diffuses faster than does Ca2+, based on a diffusion constant of ~38 μm2/s for Ca2+ and ~280 μm2/s for IP3. In this issue, Dickinson et al. (5) report that IP3 diffusion is much slower than initially thought, in the range of 5 to 10 μm2/s. This may have consequences for our understanding of how [Ca2+]i changes are propagated between cells. PIP2, phosphatidylinositol 4,5-bisphosphate; GPCR, G protein–coupled receptor.



Acknowledgments: This paper is dedicated to M. J. Sanderson, who recently passed away. He significantly contributed to our understanding of how IP3 contributes to cell-to-cell calcium signaling. Funding: The work of my group is supported by the Fund for Scientific Research Flanders, the Interuniversity Attraction Poles Program (Belgian Science Policy), and Ghent University.
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