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E-Conference: Defining Calcium Entry Signals

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Defining plasma membrane calcium channels

1 June 2004

Bernd Nilius

(a) The properties of calcium channels:

1. Do we ask the right question? The main question in this topic, “can we define plasma membrane channels mediating Ca2+ entry signals”, seems to be somewhat redundant because all Ca2+-permeable channels will mediate Ca2+ entry signals. Obviously, we will focus on “store-operated Ca2+-permeable channels” (SOCs). However, we should be aware that even a monovalent channel could couple to Ca2+ entry, e.g. in case where a “store-operated” Na+-permeable channel is tightly coupled to NCX (1). Possible candidates could therefore be searched in the whole range of cationic channels, Ca2+-permeable or even Ca2+-impermeable non-selective cation channels.

We should also be aware that manipulating “global” or “domain” [Ca2+]i will probably affect a plethora of store–dependent processes as well as Ca2+-permeable channels and we have to carefully define a feedback-independent measuring system (2). Needless to say, identifying the molecular nature of store-operated Ca2+ entry pathways will help to answer the questions how are SOCs activated.

2. What is required to define a “store-operated channel”? The main difficulty therefore is: Can properly define that activation of a Ca2+-permeable channel is indeed mediated by an intracellular Ca2+ store? Even this question has not been solved properly.

a) It must be shown that a “channel” is activated by store depletion in a native system at a physiological temperature. This should be preferentially done in a patch-clamp approach and the best would be to show a close correlation between current activation/deactivation and changes in the Ca2+ content of an intracellular Ca2+ store.

b) The most straightforward protocol would be showing activation of a whole-cell current -preferentially measured under perforated patch conditions- caused by store depletion and to show a single channel equivalent (as has been done for all voltage-operated Ca2+ channels). Under these conditions, currents through a highly selective Ca2+ channel might be difficult to dissect. In this case, non-stationary noise analysis should be used (3). In case of channels with a low permeability for Ca2+, as for most of the TRP candidates, the whole cell approach should be most straightforward and it is still surprising that this approach is widely neglected. Single channel data alone are often worthless.

c) A quantitative correlation must be shown between intracellular Ca2+ signals and SOC entry of Ca2+ taking into account Ca2+ buffering, extrusion and sequestration (4,5). This also requires that we know biophysical properties of SOC, e.g permeation and gating features, and that we know its regulation under the chosen conditions, especially modulation by [Ca2+]i.

d) For identification of a molecular candidate, the critically defined “SOC” phenomenon should disappear when the candidate protein or gene is eliminated in vivo or in a native cell. This all together has not been accomplished yet.

3. Let us assume that we have clearly identified a SOC: The question is now what should be the biophysical properties of such a channel. From the biophysical point of view, it should be Ca2+ permeable. Obviously, the best characterized store operated channel is CRAC, which has been measured in several cell types, particularly in blood cells(6). CRAC is characterized by a PCa/PNa ~ 1000, but a very low single channel conductance for monovalent cations (~0.1 pS) and in the fS range in the presence of Ca2+(7,8). So far there is no real molecular candidate for such a channel. All other channels described as SOCs only partially match the criteria defined above and the question remains, whether they are really SOCs.

(b) The role of known channel proteins: 1. Are TRPs SOCs? Most intriguingly, TRP channels have been overwhelmingly welcomed as the missing molecular candidates for SOCs(2,9). But are TRPs really SOCs? The evidence that depletion of intracellular Ca2+ stores initiates or modulates activation of various TRP channels is overwhelming. Such experiments have been shown for all TRPCs (2) and also for TRPV6 (10). TRPA1 can be activated by a store-depletion protocol but is unlikely a SOC (11). Most TRP channels have a low Ca2+ selectivity with a PCa/PNa between 0.1 and 10 (2,12) and contribute undoubtly to Ca2+ entry. TRPM4 and TRPM5 are very likely Ca2+ impermeable (13,14), however, TRPM5 has also been considered as store operated (15). Most of the studies were done in heterologous expression systems. Needless to say, we cannot rely on cell systems in which the signalling cascade between store and plasma membrane might be altered, the correct stoichiometry might be violated, or the correct players (subunits) might be missing. In addition, in any overexpression system the significance of local changes in Ca2+ concentration in a domain around the channel might be dramatic, considering the extraordinary high Ca2+ sensitivity of nearly all TRPs.

The question remains, whether TRPs fulfil the above-described criteria for SOCs. Referring to the last critrion (1d), only a few studies have been performed. In a trpc4-deficient model a SOC phenomenon disappeared (16). However, the biophysical properties of heterogeneously expressed TRPC4 channels do not match the “lost” currents (17,18). TRPC1 has been described in a knock -down approach as essential component of SOC (19). Recently, TRPC1, TRPC4, TRPV6 have all been described in native cells and using a knock-down approach they were all found to participate in SOCs, although with different modes of activation (20). Surprisingly, none of these studies uses cell models from the respective knock-out models. None of the studies, which are only a selection from many reports, clearly defines a “store-operated channels” according to the above-mentioned criteria. With no doubt, we cannot sufficiently answer this simple question yet.

2. TRPs and CRAC: The same dilemma is true for answering the question: Are TRPs CRACs? The only highly Ca2+ selective channels in the TRP family are so far TRPV5 and TRPV6 with PCa/PNa > 100 (2,21). Several features of TRPV6 (and TRPV5) are identical with those of CRAC. However, single channel conductance, open pore block by intracellular Mg2+, and permeation for Cs+ (which all reflect pore properties) and also several pharmacological properties substantially differ between TRPV6 and CRAC (22). Therefore, TRPV6 is very likely not CRAC. However, endogenous CRAC was markedly depressed by expression of N-terminal TRPV6 fragments, indicating a possible modulatory role of TRPV6 on CRAC. However, these findings underline again that TRPV6 is not CRAC (23). So far, no CRAC channel seems to be present in the TRP family, at least judged from our knowledge from expressed channels including heteromers. In any case, the superficial identification of TRPs as SOCs must be avoided.

In an alternative approach, however, we should consider that SOCs could be attributed to transporters (remember that most of the ATP driven primary transporters, many exchangers, and even ClC-ec1 (24,25), the prokaryotic predecessor of ClC channels, are electrogenic and see the very small conductance of CRAC for monovalent cations (7)). Two other exciting examples for a possible involvement of transporters in Ca2+ entry have been recently publisehd. The divalent metal transporter-1 (DMT1/DCT1/Nramp2) can easily be converted into a Ca2+ channel by a single mutation, G185R, inducing a constitutive open, highly Ca2+ permeable channel (26), and the mitochondrial Ca2+ uniporter is a highly Ca2+ permeable channel (27). We should be aware that permeation pathways are also coupled to transporters. Should these examples not already draw our attention on novel candidates?

(c) The assembly and organization of channels: Is it worthwhile discussing this issue if we cannot answer the previous question? Can the dilemma that no reliable candidate for SOC is available be solved by assuming that known channels, including TRPs, form heteromers, assemble with subunits or form signalplexes with regulatory proteins? Nobody will deny this possibilty, and from the philosophical view of Karl Popper, this question cannot be falsified. Focusing on CRAC, which is the only reliably SOC described so far, the crucial experiment will be whether the correct pore properties of CRAC will emerge in a heteromer or a signalplex. This has not yet been shown.

There is no doubt that multimerisation occurs for many TRPs, such as TRPC1/4/5, TRPC3/6/7, TRPV1/3, TRPV5/6, TRPM4/5, TRPM6/7 and that heteromer formation changes permeation and kinetic properties of those channels. Partnership with other proteins acting in a signalling network has been most impressively shown for Drosophila TRPs (28). So far, various modulators of mammalian TRPs have been identified, including calmodulin, the inositol (1,4,5) trisphosphate receptor, ankyrin, NHERF, PI3-kinase, annexin 2, S100A10, caveolin-1, 80KH, PLCg, TrkA, RGA, MAP7, which probably act by direct binding to TRPs (2,12). The most intriguing assembling function might be credited to the adapter protein homer, which couples TRPC1 to the IP3R. Disassembly of the TRPC1-homer-IP3R complex parallels TRPC1 activation (29). However, even this example provides more evidence for TRPC1 modulation by protein-protein interaction than for TRPC1 being a SOC.

Again, the bottom line, none of these many interactions described so far in the TRP family has solved the SOC dilemma. Again, we have to be open looking beyond the now somewhat absorbing TRP hypothesis. Then the question remains, what else, if not TRPs?


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