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E-Conference: Defining Calcium Entry Signals

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How many SOCs are there?

1 June 2004

Reinhold Penner

Forum discussions often benefit from an initial controversial "spark". So let me make a provocative initial assessment that relates to the question "How many store-operated currents/channels are we dealing with?"

My provocative hypothesis would be that, although there is an abundance of papers that use the term store-operated for various (mostly non-selective) currents and channels, IMHO so far only one current has been established beyond reasonable doubt as store-operated and that would be Icrac (Calcium Release-Activated Calcium-selective current).

I do not wish to be misunderstood. I do not exclude the possibility that other store-operated channels exist. However, none of the non-CRAC conductances have been subjected to the same rigorous experimental tests from a number of independent labs that establish the final activation mechanism as unambiguously store-dependent. So what are the criteria one might want to see fulfilled for a genuine store-operated current?

1. Obviously, there has to be a current that can be measured directly with electrophysiological techniques. Fura-2 experiments or pharmacological manipulations are indirect and not sufficient to demonstrate a store- operated current (even if the cell under investigation has been demonstrated to possess a store-operated current).

2. Independent and unrelated store-depletion protocols should all lead to non-additive recruitment of the same conductance (e.g., IP3, BAPTA, ionomycin, thapsigargin, receptor agonists). Either alone is not sufficient to establish store-dependence.

3. While cell lines can be good models for store-operated mechanisms, it is imperative that a store-operated mechanism is also found in native cells the cells are derived from.

4. Although it is conceivable that calcium-impermeable channels could be triggered by store-depletion, the ultimate outcome is likely calcium entry and the current should have some calcium permeability if not high selectivity for the ion.

5. The currents should activate in a calcium-independent manner. That is unbuffered intracellular solutions should not lead to activation of SOC and mechanisms that prevent store-depletion (e.g. heparin or IP3 antagonists should prevent SOC activation without blocking the channels directly, whereas non-IP3-dependent store-depletion protocols such as ionomycin should still function under those conditions).

So, from my point of view, here is an ideal experiment that would go a long way to establish a SOC: In a whole-cell experiment, perfuse a cell with [Ca]i buffered slightly above resting levels (say 200-300 nM using 10 mM BAPTA/ EGTA, add physiological MgATP and ideally K+ as physiological cation). In the absence of a store-depletion stimulus in control cells, this should cause no SOC activation for 10 min of whole-cell recording. Then perform the same experiment and induce store-depletion after 3-5 min by at least three different protocols (e.g. brief ionomycin application, receptor agonist, release of caged IP3). All of these stimuli should cause a rapid and supramaximal SOC current. Finally, apply all three stimuli together (or better sequentially) to demonstrate that they all converge on the current in a non-additive manner.

6. Heterologuous expression or knock-out systems are dangerous for several reasons. Compensation mechanisms, up- or downregulation of endogenous SOC, aberrant signaling, reorganization of signaling mechanisms are all possible caveats to keep in mind when interpreting results from such systems.

So in summary, I believe that it is possible to activate a number of channels following store depletion, but the crucial question as to whether the channels are truly store-operated is difficult to answer unless many different types of experimental conditions are tested. A case in point that illustrates this problem is the fact that TRPM7 channels (termed MagnuM for Magnesium- Nucleotide regulated Metal ion currents in native cells) have been mistaken as CRAC channels, simply because those channels are activated under the very same experimental conditions that were previously considered to specifically only activate CRAC channels. It takes some effort to dissociate these conductances even under controlled patch-clamp conditions. Another case in point is that of TRPV5 (ECaC) and TRPV6 (CaT1), which both exhibit a great degree of similarity to CRAC channels, yet on close scrutiny are clearly distinct and not store-operated.

I hope that these thoughts have stirred the pot and I would like to hear some opinions about which channels other than CRAC the forum members consider as established store-operated channels or which are close to being classified as such.

Aloha, -Reinhold


Reinhold Penner MD, PhD Center for Biomedical Research The Queen's Medical Center & University of Hawaii 1301 Punchbowl St. - UHT 8 Honolulu, HI 96813

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