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E-Conference: Defining Calcium Entry Signals

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The importance of terminology and electrophysiology

3 June 2004

Trevor Shuttleworth

I think it is clear that the field is suffering under a profound confusion in terminology – a fact that was probably a major driving force for initiating this forum.

In some areas, terminology can devolve into arcane semantics, but this is certainly not the case for Ca2+ entry channels where often the same term is used for obviously widely different phenomena. Part of the problem is that we are often trying to define a channel, or at least a Ca2+ entry pathway, by techniques that are really hopelessly inadequate to make such definitions. Reinhold and Bernd have already alluded to this in their postings.

The use of changes in cytosolic Ca2+ based on fluorescence measurements is simply not suitable to define a channel. Similarly, all the pharmacological tools currently available for such channels are so poor that they cannot be used with any confidence to discriminate between different pathways for entry.

The only way we can even begin to characterize the pathways operating under various conditions in any given cell type is by measuring the channel properties themselves using electrophysiological techniques. However, even this is not without significant problems. Because it appears that many of these Ca2+ entry channels have such small conductances, almost all studies have to use whole-cell techniques, with the associated problems of contamination from other currents etc. that has already bedeviled the field.

So, based on what we know, what criteria can we use to define these Ca2+ entry pathways?

First, we know something, but not nearly enough, about their Ca2+ selectivity. Thus, there are entry channels that, under normal conditions, show a high selectivity for Ca2+ [e.g. CRAC (Ca(2+) release-activated Ca2+ channels) and ARC (arachidonate-regulated Ca2+ channels)], in contrast to others that are essentially nonselective cation channels. Although both can contribute to overall Ca2+ entry, one important difference is that a major physiological effect of the latter will be to depolarize the cell. This could have an important consequence for Ca2+ signaling in that it would result in the opening of voltage-gated Ca2+ channels in those cell types that possess such channels (e.g. smooth muscle cells).

The real problems arise when we try to use the mode of activation as a criterion for classification – because even our basic understanding of these processes is so poor. Most importantly, the direct means of activation is almost always unknown.

There do seem to be channels that are activated only as a consequence of store depletion, and others that are activated only as a result of receptor activation. The key word here, in each case, is "only". Obviously, any member of the first group could be activated by receptor activation, providing the receptor couples to the generation of IP3 (or some other store-depleting messenger) and that this is produced in sufficient quantity to effect an adequate store depletion. However, these should be distinguished from those channels whose activation is solely dependent on receptor activation and is entirely independent of store depletion. An appropriate test here might be whether the channel activity can be increased by appropriate receptor activation after maximal depletion of the intracellular stores.

As to the role of known channel proteins – here we are obviously talking about the TRP family, as these are currently "the only game in town". In this, I will just focus on the TRPC family.

Reinhold and Bernd have already alluded to the many potential problems of expression and knock-down approaches to the study of these proteins. However, to me, the fundamental problem with the "TRPC hypothesis" is that it is clear that, in many cell types, the endogenous Ca2+ entry channels – both store- operated, and noncapacitative – are of the highly Ca2+-selective type (see above). As such, none of the known TRPC family members fit the bill as candidates for these channels.

For some time now, the standard argument has been that the TRPCs form heteromultimers that may possess unique characteristics that differ from either, or any, of their individual components. However, Ca2+ selectivity is likely to be a fundamental property of the channel pore itself, and none of the few published reports on such TRPC heteromeric channels show the required levels of Ca2+ selectivity. It seems curious, given the widespread use to the "heteromeric channel" argument, that so few studies have appeared where such channels have been investigated.

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