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E-Conference: Defining Calcium Entry Signals

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How many stores are there?

7 June 2004

Reinhold Penner

I would like to raise the issue about "how many stores are there?". I believe that this is a confusing issue and directly relates to some of the points raised by Mike Berridge and Trevor Shuttleworth when they talk about physiological concentrations of agonists.

I think it is safe to say different agonists are capable of generating different Ca2+ signals even in the same cell. Moreover, these signals change as a function of agonist concentration even for the same agonist. Furthermore, we must consider different "receptor concentrations", since receptors may not necessarily be expressed in equivalent numbers.

To complicate matters, it is also clear that we cannot regard the ER as a single homogenous store, since store heterogeneity has been observed in numerous cell types. If we then further consider that receptors and stores may have specific spatial arrangements, which would certainly have important consequences for downstream second messenger production, we can easily envisage a complex situation that defies simplistic interpretations.

Nevertheless, I'd like to point out a few results we have obtained in assessing some of the intricacies of SOC activation in RBL (rat basophilic leukemia) cells. These cells, just like Jurkat T cells (the other well-established model system for studying CRAC), offer some advantages to at least appreciate these problems. In these cells, we know that agonist stimulation in patch-clamp experiments activates a single Ca2+ entry pathway, namely Icrac (yes, there could always be others, but they simply do not show up even under the most physiological experimental conditions that patch-clamp recordings can provide).

With this in mind, we can try to assess the dose-response relationship for Icrac activation by intracellular IP3 concentrations. We have shown that this relationship is rather steep and proceeds essentially in an all-or-none manner, with IP3 concentrations of 3 micromolar or more required to trigger Icrac. When we assess the store release by IP3 under similar experimental conditions, IP3 concentrations of 1 micromolar or less already empty the bulk of IP3-sensitive stores, without triggering significant CRAC. This has prompted us to propose that CRAC channels are under the control of functionally (and possibly physically) distinct "CRAC stores" that do not contribute significantly to cytosolic Ca2+ release transients.

Obviously, perfusing IP3 into a cell via the patch pipette is not how IP3 is generated physiologically by receptor agonists. This process occurs at the plasma membrane and it is therefore obvious that IP3 concentrations are highest underneath the plasma membrane, which is also likely to be the location of the "CRAC stores". So how can we link store release and CRAC activation at different agonist concentrations? I would like to propose the following scenario and am happy to receive flames ;-)

1. Very low, subtreshold agonist concentrations: Very low carbachol concentrations (30 nM) in RBL cells will not trigger a visible Ca2+ release transient and will not activate CRAC. This does not mean that signaling or IP3 production do not occur, since giving a second identical subthreshold stimulus within a specific time window will produce a Ca2+ influx without generating a Ca2+ transient.

We believe this reflects the activation of CRAC by localized IP3 increases triggering the CRAC stores. The first subthreshold stimulus acts as a priming step, which then changes the apparent sensitivity of the CRAC stores by inhibiting the IP3-metabolizing enzymes that normally keep IP3 levels low around the CRAC stores (presumably via IP4-mediated inhibition of IP3 5-phosphatase). The second stimulus can then reach the IP3 threshold required to activate CRAC. But the process is very slow and requires perfect timing of agonist stimulation.

So, Ca2+ entry through CRAC appears to occur at very, very low agonist concentrations. Such low concentrations of agonis do not trigger a bulk ER release transient, because IP3 remains localized below the plasma membrane. The IP3 never reaches the deep cytosolic ER to generate a significant Ca2+ release transient.

2. Low agonist concentrations: These are presumably the ones that Mike and Trevor allude to as "physiological". Here the cell responds to low "above- threshold" agonist concentrations by generating single or repetitive Ca2+ transients, but without necessarily producing a significant plateau response. Surely, it seems surprising that the plateau phase does not show up when even subtrheshold agonist concentrations can lead to a detectable calcium transient (see point 1 above). Well, one could interpret this as a cross talk between release, uptake, and influx. I would hypothesize that the low agonist concentrations produce enough IP3 to reach both the CRAC stores and the deep ER. Since the ER is more sensitive to IP3 (possibly due to less IP3 metabolism and/or different IP3 receptors with higher sensitivity towards IP3), it can release Ca2+ very quickly at submicromolar IP3 concentrations, thereby generating the Ca2+ transient.

Although the CRAC stores may also be initially depleted by the above- threshold stimulus, they could immediately refill (possibly due to higher pump rates into a smaller-volume storage compartment). This may rapidly terminate the ongoing slow activation of CRAC channels, curtailing any development of a plateau phase.

3. High agonist concentrations: Here both stores are maximally and rapidly depleted and neither IP3 metabolism nor uptake mechanisms can counteract this, giving rise to the typical biphasic signals (Ca2+ transient followed by influx plateau).

So I think that store-operated influx could potentially occur at all levels of agonist stimulation and is not necessarily a feature of high (or even non- physiological) agonist concentration. The specific circumstances then determine how much of it we actually see reflected in the overall Ca2+ signal. If we accept that a specialized CRAC store exists, which may not even generate much of a release transient, then we face even more challenges in evaluating fura-2 signals...

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Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882