Processes that mediate activation of calcium entry channels

5 June 2004

James W Putney

What are the physiological transduction processes that mediate activation of Ca2+ entry channels?

In my opinion, regulated Ca2+ channels consist, as far as we know, of two basic types: Those which are gated by the binding of ligands, and those which are gated by physical forces.

The latter would include voltage-gated channels, stretch- or contact-activated channels (perhaps), and channels gated by changes in temperature. The ligand-gated channels include those gated by actions of extracellular ligands or intracellular ligands. The intracellular ligands may be ions, small molecules or second messengers, or proteins or, more likely, specific sequences within proteins.

Although we do not know the mechanism of gating store-operated or capacitative calcium entry channels, it is presumed that they are ligand-gated in some manner, either by a diffusible mediator or by another cellular protein. The physiological mechanisms and contexts for activation of channels regulated by physical forces is straightforward, as is the case for those gated by extracellular ligands. The chairs of the conference have made it clear that the current discussion is to be focused on the mechanisms of activation of channels gated by intracellular ligands, whether store-operated or otherwise, the modes of interactions between these pathways, and the physiological relevance of these different mechanisms of channel regulation.

(a) The role of Ca2+ stores in activating entry:

Are changes in intracellular stores necessary or sufficient (or both) to activate Ca2+ entry? Are intracellular Ca2+ stores physiologically depleted under normal cellular conditions?

The easiest part of this question is: Are changes in intracellular stores sufficient to activate Ca2+ entry? The clear answer to this is yes, as demonstrated by any number of strategies, including discharge of stores with messengers such as IP3, passive depletion of stores with SERCA inhibitors like thapsigargin, or passive depletion of stores with Ca2+ chelators. This ability to activate entry channels simply by depleting Ca2+ from intracellular stores defines the store-operated or capacitative pathway. This mode of Ca2+ entry activation is the most pervasive mechanism known, and as far as we know is present in all eukaryotes (although not necessarily in every cell type in multicellular organisms).

Speculatively, this mechanism may have evolved to support the requirement for Ca2+ within the endoplasmic reticulum for normal protein synthesis, folding and trafficking, and may have been exploited later as a means of generating or supporting Ca2+ signals. Since we have not as yet identified these channels at the molecular level, it is more difficult to say whether or not depletion of stores is necessary. That is, we cannot definitively rule out activation by other signaling mechanisms of the channels which are apparently store-operated. In one recent report on smooth muscle cells, store-operated channels were identified which could be activated by protein kinase C in the absence of store depletion. A major difficulty is that we do not know with certainty the downstream events following Ca2+ store depletion, and so we must entertain the possibility that these undisclosed steps could be activated or regulated by other means.

As to whether intracellular Ca2+ stores are physiologically depleted under normal cellular conditions depends upon one's definition of "normal" cellular conditions. Clearly, neurotransmitters that activate receptors coupled to phospholipase C in many instances activate store-operated Ca2+ entry mechanisms. In many experimental situations, supramaximal concentrations of agonists are used to generate maximal release of Ca2+, which in turn generates maximal activation of store-operated entry. Physiologically, it is unlikely that these saturating concentrations of neurotransmitters ever occur, and receptor signaling mechanisms probably operate in the linear range, below the binding Kd. Although this is the subject of some debate, it does appear that in many cases a close almost stoichiometric relationship exists between the extent of Ca2+ release and the extent of activation of store-operated entry. Thus, it is likely that whenever sufficient IP3 is generated to cause significant release, no matter how small, a corresponding activation of 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 which would engender far less debate among scientists in the Ca2+ signaling field.

An important case in point is the situation 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 a variety of cell types is very strong. Because the duration of these release events is brief, and the extent of Ca2+ loss is small, it is difficult to demonstrate directly that store-operated entry accompanies Ca2+ oscillations. Yet the oscillations quickly run down in the absence of extracellular Ca2+, indicating a need for Ca2+ from the outside for maintenance of the releasable stores. In instances in which this run down process was compared to maneuvers that inhibited store-operated entry, it appeared that indeed it was the store-operated entry of Ca2+, which served to support the pools of Ca2+ required for oscillations.

(b) Distinctions between receptor-operated and store-operated Ca2+ entry:

Do receptors activate distinct Ca2+ entry events from those stimulated by store-emptying? Are the Ca2+ entry events activated by receptors and emptied stores independent or integrated?

The arguments in the preceding section are not intended to imply that every phospholipase C-linked receptor activates Ca2+ entry solely by the store-operated pathway. A case in point is the TRPC channels which, at least when ectopically expressed in cell lines, can be activated in a phospholipase C-dependent manner often independently of store depletion. And in such instances, the channels can be activated by very low concentrations of agonists that do not appear to cause significant Ca2+ release.

There are clear cases in the literature of specific cell lines that utilize endogenous non-store-operated pathways for Ca2+ entry, and, in many instances, these pathways have properties similar to those which we expect from TRPC channels. A strong case has be made for such a signaling mechanism in smooth muscle cells. On the other hand, a number of studies have indicated that in T-lymphocytes the store-operated pathway constitutes the major, if not exclusive, route for activated Ca2+ entry that is essential for T-cell activation. The extent to which different mechanisms account for the physiological regulation for Ca2+ entry in other cell types has not been extensively investigated, but such studies are clearly needed.

Are the two mechanisms independent or integrated? Little work has focused on this issue, but in a few studies a somewhat reciprocal relationship between the two pathways was demonstrated. That is, there appeared to be mechanisms, not always clearly defined, through which the activation of one pathway inhibited concomitant activation of the alternative pathway.

(c) The role of intracellular Ca2+ release channels:

Do InsP3 receptors or ryanodine receptors, or both, mediate or modify Ca2+ entry events?

I can only give a rather short answer to this question. There is fragmentary evidence in the literature for regulation of store-operated channels by IP3 receptors or ryanodine receptors, and at least one report of the regulation of non-store-operated channels by IP3 receptors. However, some of these findings have not be readily reproduced in other laboratories. I think it is fair to say that a consistent pattern of behavior or mechanism has not developed as yet. Additional studies are needed to assess the roles of intracellular release channels in various Ca2+ entry mechanisms.

(d) The function of mediators and adaptors in Ca2+ entry:

What role is played by structural or adaptor proteins (including Homer, NHERF, InsP3Rs, and PLC-gamma), or by soluble cellular messengers (such as InsP3, cyclic nucleotides) and lipid messengers (including diacylglycerol, arachidonic acid, and PIP2)?

This question seems to be an expanded version of the one above. Again, some very interesting studies have been published implicating each and every one of the listed regulatory substances, but in most cases a well-established and general paradigm of mechanism has not been established. One might also include the elusive calcium influx factor and lysolipids in the list of putative mediators for which there is encouraging, but as yet limited, published support. Exceptions would be the cyclic nucleotides and diacylglycerol, and possibly arachidonic acid, for which a growing body of evidence supports their roles as regulators of specific classes of Ca2+-permeable channels.