Perspective

Store-Operated Calcium Channels: How Do We Measure Them, and Why Do We Care?

See allHide authors and affiliations

Science's STKE  27 Jul 2004:
Vol. 2004, Issue 243, pp. pe37
DOI: 10.1126/stke.2432004pe37

Abstract

A major mechanism whereby calcium entry into cells is regulated is the store-operated or capacitative calcium entry pathway. In this article, two basic issues are discussed: (i) the methods investigators use to measure store-operated entry, and (ii) the role played by the store-operated pathway in responses to hormones and neurotransmitters under physiological conditions. The two topics are considered together because they are closely interrelated; as we begin to ask questions about calcium movements at low concentrations of agonists, the technology to measure these movements becomes increasing challenging.

Plasma membrane calcium channels fulfill a multitude of purposes, including providing rapid signals for cell activation, providing delayed signals regulating the growth and differentiation of cells, and maintaining intracellular calcium homeostasis. Certain calcium entry channels appear to function, at least in part, to maintain a relatively constant concentration of calcium in the endoplasmic reticulum (1, 2). These channels have been designated "store-operated channels" or "capacitative calcium entry channels." Although we often speak of them as calcium entry channels, in very few cases have the single channels responsible for capacitative calcium entry actually been observed; rather, the case for such channels is an indirect one that is based on a combination of fluorescence measurements of transmembrane Ca2+ fluxes and electrophysiological measurements of transmembrane currents. The concept of capacitative calcium entry is now 18 years old (3), and in 1992 the first report of a store-operated current appeared, the now extensively studied and well-characterized calcium release-activated calcium current, or ICRAC (4).

Methods of Studying Store-Operated Entry

Most studies investigating store-operated entry use either (or both) of two basic methods: measurement of concentrations or fluxes of Ca2+ or other divalent cations with one of the fluorescent (or sometimes bioluminescent) Ca2+ indicators (5), or electrophysiological measurement of transmembrane fluxes of ions, usually with the patch-clamp technique (6). Certainly the former technique is by far the more frequently encountered, probably because of the greater technical ease of measuring fluorescence compared to measuring the very small currents associated with store-operated channels, and because of the speed with which data can be accumulated on large numbers of cells. However, the reporting of the intracellular concentration of free calcium ([Ca2+]i) as measured with Ca2+ indicators can be modified by a number of parameters that are unrelated to store depletion and are sometimes not adequately controlled. The most common of these are effects of Ca2+ buffering or removal mechanisms and of membrane potential, which determines the driving force for Ca2+ entry. The former is most readily dealt with by use of surrogate ions that will pass through store-operated channels and excite (or sometimes quench) fluorescent indicators. Ba2+ is often used, but Ba2+ can have pharmacological effects of its own. Mn2+, which quenches some of the Ca2+-indicator dyes, can also be used. Ba2+ and Mn2+ have the additional advantage that they do not generally activate Ca2+-dependent processes, avoiding the possibility of secondarily recruiting Ca2+-activated channels. Sr+ is sometimes used because it helps to distinguish among certain channels types, but Sr+ is a substrate for Ca2+ transporters and buffers and will mimic Ca2+ in activating most Ca2+-dependent responses. Membrane potential can be controlled fairly well by chemically clamping to ~0 mV with an extracellular solution containing K+ ions substituted for Na+. Even better, fluorescent measurements can be made together with the patch-clamp technique to achieve more defined and rigorous control of membrane potential.

In the latter case, the question might be asked, if one is going to the bother to patch-clamp a cell, why not just measure the current? In fact, in the ideal situation, that is exactly what should be done; current measurements are inevitably better controlled and less ambiguous than fluorescence measurements. However, in some cases the currents underlying store-operated entry are vanishingly small and difficult to study in a meaningful way. However, they are almost always readily revealed by use of fluorescent indicators. Thus, despite all of the drawbacks outlined above, fluorescence measurements do have one distinct advantage over current measurements: They are considerably more sensitive and can detect divalent cation movements under conditions (that is, often more physiological conditions) in which current measurements are not practical.

Functions of Store-Operated Channels (Why We Care)

Regulation of Ca2+ entry by intracellular stores is a widespread phenomenon, present, as far as we know, in all eukaryotes from yeast to man. As such, it is interesting that store-operated channels appear to predate the signaling function of inositol 1,4,5-trisphosphate (IP3) with which they are so often associated. It is possible that store-operated entry evolved as a means to ensure a relatively constant concentration of Ca2+ in the endoplasmic reticulum, where it is required for proper protein synthesis and processing (7). However, it is clear that these channels have evolved to take on other cellular functions--specifically, in the generation or support of cytoplasmic Ca2+ signals in response to neurotransmitters and hormones (810). Store-operated channels most typically act in concert with the phospholipase C (PLC)–IP3 signaling pathway, but other means of intracellular Ca2+ discharge can also activate store-operated entry [for example, see (11)]. The evidence is overwhelming that this mechanism has an important role in cellular Ca2+ signaling and homeostasis under physiological, as well as pathological, conditions [see (1), and multiple reports and reviews cited therein]. The nature of the Ca2+ signals in various cell types varies considerably. In lymphocytes, for example, Ca2+ stores are relatively small, the capacity for entry through the plasma membrane is large, and thus entry often dominates the receptor-linked calcium signals. In epithelial cells, on the other hand, Ca2+ stores are large, and release from intracellular stores appears to be the predominant mode of signaling. In the latter case, and with low, physiological concentrations of agonists, this release typically occurs in the form of periodic transient release events termed Ca2+ oscillations (1214). It is clear that these oscillations require Ca2+ entry for their maintenance and it has been generally supposed (but see below) that this entry is store-operated. It has also been suggested that store-operated entry might play a more direct role in generating or modulating the oscillations by contributing Ca2+ to the process of IP3-sensitized calcium-induced calcium release (1517).

Other Modes of Ca2+ Entry

There is evidence for the existence of Ca2+ entry mechanisms that are regulated by membrane receptors, often PLC-linked receptors, but which do not depend on store-depletion (1820). Interestingly, and perhaps not surprisingly, these mechanisms seem to be most important for agonist concentrations below those which cause detectable release of calcium from intracellular stores. Although the molecules that make up these channels are not known with certainty (nor, for that matter, are they known for the store-operated channels), a family of ion channel subunits, the transient receptor protein C (TRPC) channels, have the properties of being activated at lower agonist concentrations than are store-operated channels, by a signal linked to PLC (21). A strong case can be made for the physiological importance of such channels in certain cell types, smooth muscle being a particularly good example (8, 22). However, it is not clear how pervasive these non-store-operated mechanisms are. Nonetheless, it has been suggested that under physiological conditions, these might be the only or predominant mechanisms for receptor-regulated entry (23, 24). Thus, it is important to examine more closely the role played by store-operated channels under physiological conditions.

Although this is the subject of some debate, it appears that in some cases a close, almost stoichiometric relationship exists between the extent of Ca2+ release from intracellular stores and the extent of activation of store-operated entry (21). Thus, it is likely that whenever sufficient IP3 is generated to cause significant release, no matter how small, a corresponding activation of Ca2+ entry will occur. Therefore, the question of the physiological relevance of the role of store-operated channels is tied in a way to the question of the physiological role of IP3-induced Ca2+ release, an issue that would engender far less debate among scientists in the Ca2+ signaling field. An important case in point is the situation, discussed above, in which the [Ca2+]i in cells oscillates through a mechanism involving cyclical IP3-induced Ca2+ discharge. The case for the physiological relevance of Ca2+ oscillations to the fundamental processes of Ca2+ signaling in various cell types is very strong. The duration of these release events is brief, and the extent of Ca2+ loss is small, such that the predicted entry of Ca2+ is correspondingly small. For this reason, it is difficult to demonstrate directly that store-operated entry accompanies Ca2+ oscillations. Yet the oscillations eventually cease in the absence of extracellular Ca2+, indicating a need for Ca2+ from the outside for maintenance of the releasable stores. This situation is very different from the one in which agonist concentrations are below the threshold for activating release; in an oscillating cell, clearly release is occurring, albeit briefly. There is no rise in baseline [Ca2+]i between the spikes, so the entry is very small. Calculations of the amount of current associated with even modest steady-state rises in [Ca2+]i indicate that such currents, even when there is a rise in baseline [Ca2+]i, are at the threshold of detection (25). The prediction is that any influx-related current associated with [Ca2+]i oscillations will be undetectable. (Parenthetically, one could argue that any Ca2+-selective current that can be measured under such conditions is paradoxically large.) Thus, we are forced to address the issue of signaling mechanism or channel type by more indirect means.

One approach that has been traditionally fruitful in the signal transduction field is the use of pharmacological tools to identify specific molecules or pathways. Unfortunately, we do not have a single, highly specific, and potent inhibitor of store-operated channels, such as the various channel-specific neurotoxins that act on voltage-regulated channels. However, a survey of the literature indicates that a complete block by both very low(1 μm and below) concentrations of lanthanides together with block by the drug 2-aminoethoxy diphenylborane (2APB) in the 20 to 50 μM range is diagnostic for store-operated channels (26). In a detailed study of [Ca2+]i oscillations in the classical model for this process, the hepatocyte, Gd3+, 2APB, and a number of other inhibitors known to block store-operated entry, all inhibited [Ca2+]i oscillations at the expected concentrations (27). Inhibitors that block non-store-operated channels, for example, LOE-908, were without effect. These findings strongly implicate store-operated channels as the basis for sustenance of [Ca2+]i oscillations in hepatocytes. Similar pharmacological studies will be needed to determine the pathways involved in the responses to low, physiological concentrations of agonists in other cell types.

Conclusion

In this somewhat eclectic Perspective, I have tried to make two general points that I believe are related. The first has to do with measurements of store-operated fluxes of Ca2+. Although electrophysiological measurements are by far the preferred method, investigations of store-operated entry in many systems, and possibly in all systems at low levels of stimulation, require the use of more sensitive fluorescent indicators. This quandary of methodology is an important factor in the currently debated question of what entry mechanisms are the most significant in various biological systems under physiological conditions.

Reports documenting the involvement of store-operated channels in various physiologically important processes are legion. By contrast, reports of the involvement of more recently discovered non-store-operated pathways are much fewer in number. More importantly, it does not appear that a consensus across different laboratories has yet been reached as to how these alternate pathways operate, or, in some cases, whether they even exist. But it is early days, and we can look forward to a better understanding in the future of the physiological roles of store-operated and non-store-operated channels, and possibly their interactions with one another.

Editor's Note: This Perspective is part of a series related to an E-Conference held at Science's STKE. Invited participants were asked to provide Perspectives that summarized the ideas, conclusions, areas of controversy, and challenges for future work that emerged in the online discussion at http://stke.sciencemag.org/cgi/forum-display/stkeforum;14.

References

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
View Abstract

Stay Connected to Science Signaling

Navigate This Article