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E-Conference: Defining Calcium Entry Signals

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Processes that mediate activation of calcium entry channels

5 June 2004

Donald Gill

The terms "store-operated channel" and "capacitative Ca2+ entry" are becoming increasingly familiar in the literature and are finding their way into the vocabulary of an ever greater diversity of papers. Thus, investigators in quite different fields may feel confident that Ca2+ signals in their particular cell types are mediated by the activation of store-operated channels. However, those of us studying this process are becoming increasingly unsure about the basis of store-operated Ca2+ entry, that is, both the signals that generate such entry and the channels that mediate it. It was this disparity between the increased acceptance of the concept of store-operated channels and the decreased understanding of the function of store-operated channels that prompted Randen and I to instigate the E-Conference. This was to allow experts and other interested investigators to air their views in an uninhibited yet highly visible forum.

A major focus of this E-Conference is to highlight the distinction between physiological receptor-activated Ca2+ signals and what we frequently study as "store-operated" Ca2+ signals. The latter are generated mostly by a nonphysiological emptying of stores using ionophores, SERCA blockers, or infusion of InsP3 into the cells. Increasingly, we learn that the substantial depletion of stores may be a nonphysiological event. An exception to this may be small blood cells (such as lymphocytes) with large nuclei and limited cytoplasm, in which activation of PLC-coupled receptors may cause a significant decrease in luminal Ca2+. In most other cell types in which the endoplasmic reticular network is extensive and pervasive, the capacity of Ca2+ within the lumen is great and luminal depletion in response to physiological agonists may be very small.

There are a number of known channel proteins that have been suggested to function in a "store-operated" mode. Notable among these candidates are the TRPC channels denoted as being the mammalian TRP channels most similar to the original TRP channels that mediate Drosophila phototransduction. There seems little question that the TRPC channels are receptor-operated. Indeed, the rhodopsin/G protein/PLC photosignaling system of fly retinas is a prototype for such signaling. At least three closely resembling members of the mammalian TRPC family (TRPC3, TRPC6, and TRPC7) can be activated by the PLC product, diacylglycerol (DG) and it would appear that this PLC product could explain activation of these channels.

However, in fly retinas, the TRP channels present do not appear to be activated by DG. Moreover, other mammalian TRPC channels (including TRPC1, TRPC4, and TRPC5) are not activated by DG. Yet, somewhat enigmatically, when expressed in cells, all of these TRPC channels can be activated by PLC-coupled receptors in what appears an almost identical fashion to the TRCP3/6/7 family.

Strangely, we find that in A7r5 smooth muscle cells endogenously expressing high levels of TRPC6 channels, there is little activation of these channels upon addition of DG. In contrast, in the same cells, exogenously overexpressed TRPC6 is activated by DG. Are we therefore to believe that physiologically the TRPC family of channels respond to a common alternative PLC-derived signal? One such signal could be a decrease in PIP2, but, while this is an attractive hypothesis, there is no direct evidence for this action at present.

We are starting to believe that the transduction mechanism for store-operated channels is perhaps a more "general" phenomenon rather than a highly specific and directed signal. Whereas the conformational coupling model has been an attractive scheme and has derived some support from the possible role of InsP3Rs, we consider that the analogy of this model with the functioning of the triad junction in skeletal muscle is somewhat flawed. Thus, many believe the simple direct interaction between a Ca2+ entry channel and a Ca2+ release channel may be a universal paradigm for Ca2+ signals in all cells.

In reality, however, the triad junction in muscle serves as an exceedingly rapid means for sensing and transducing the electrical signal in the plasma membrane into a Ca2+ release signal from the SR. In contrast, the store-operated signal takes many seconds or even minutes to develop and is, hence, unlikely to require a similar rapid and direct coupling process.

The role of the InsP3R in mediating the store-depletion signal to activate store-operated channels has been a popular area of investigation and has received considerable experimental support. Nevertheless, the complete elimination of InsP3Rs through knockout procedures results in little influence on the activation of store-operated channels. Although apparently not necessary for the coupling process, it does appear that InsP3Rs may influence coupling and hence may have a regulatory rather than obligatory role on the activation of SOCs.

Whether direct conformational coupling is involved or not, our own experiments and those of others indicate that it is likely that close interactions between the plasma membrane and ER membrane are required for activation of the "store-operated" coupling process. Thus, we consider it likely that there is a spatially directed "area of influence" between of the plasma membrane and the ER. This may be through areas of close association between the ER and plasma membrane (PM), which have been observed to exist in many cell types. However, these "junctional" structures have proven extremely elusive. In most cells they are sparse at best and there is no direct evidence that they are the sites at which Ca2+ entry signals are activated.

Given that TRPC channels are interesting candidates for the process of receptor-induced signals and given the numerous reports suggesting that these channels are influenced by store-emptying, it appears that they may at least be components of store-operated Ca2+ signals in some cells. However, we are developing a somewhat different perspective on the whole process.

We are realizing that TRPC channels (as well as other TRP channel members) are channels receiving multiple inputs. We think of them as "integrators" of a number of signals, their opening reflecting the summation of multiple distinct signals. Thus, the TRPC channels can be modified by diacylglycerol (and perhaps unsaturated fatty acids), by InsP3 receptors (and perhaps ryanodine receptors), by adaptor proteins such as homer, NHERF and PLC-gamma, by protein kinase C, and by Ca2+ itself. By analogy with the TRP channels of fly retina, it would appear that the mammalian TRPC channels function within domains containing numerous structural, adaptor and modifier proteins, as well as the receptor/G protein/PLC proteins transducing the agonist-induced signals.

We would like to think that this structural domain is also a site "under the influence" of the filling status of Ca2+ stores. This would be one further input. Under conditions in which stores are totally emptied, albeit an unphysiological circumstance, then the store-derived signal may become a dominant one. Under conditions in which physiologically activated receptors are inducing store-release, small changes in store-Ca2+ may be sufficient to subtly modify the TRPC channels in a way in which larger sustained entry of Ca2+ may occur, sufficient, for example, to increase the frequency of Ca2+ oscillations.

This being said, we really do not know what this store-derived signal is. If areas of close association are the communicating sites betweens stores and PM, then it is possible that locally released factors may be mediating the communication process. In this case, we may be hard-pressed to identify and measure such mediators. However, by carefully dissecting the "tool-box" of TRPC modifiers and by changing the function of these modifiers by a combination of pharmacology and knock-down strategies, we may be able to begin to pinpoint the nature of this elusive coupling mechanism.

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