E-Conference: Defining Calcium Entry Signals
Calcium entry at low agonist concentrations
18 June 2004
1. This is obviously difficult to answer categorically, and of course my opinion is probably not entirely objective! So let me try to just give you the information.
So far, we have identified ARC currents in HEK293, HeLa, COS, CHO, RBL, and DT40 cells. It is also in parotid acinar cells (mouse and human).
In the cells we use most (the HEK 293 cells stably transfected with the m3 muscarinic receptor), we begin to see Ca2+ signals in intact cells at around 0.2 uM CCh. These are typically small, rather irregular, oscillations that are often not sustained.
At this same concentration we can measure a detectible ARC current in whole-cell patch recordings equivalent to around 40-50% of maximum ARC current. An example of these data was published in Biochem Soc Trans. 31: 916-919. Of course, AA-activated Ca2+ entry has been demonstrated in a wide variety of cells, but this may not always involve ARC channels.
Re. the "ying-yang"..... our proposal for the reciprocal regulation of ARC and SOC channels is clearly supported by a wealth of evidence from the m3-HEK cell system. But it is also consistent with demonstrations that Ca2+ entry at low agonist concentrations is entirely (or, at least, predominantly) dependent on the generation of AA in several cell types.
It also explains why the original work from Jim Putney's lab (Takemura et al) failed to detect any additional route of agonist-activated Ca2+ entry in intact cells after thapsigargin treatment. This was critical in leading Jim to propose that there was only one type of entry, i.e. capacitative entry.
As to the "preactivation" question, if you are referring to the type of protocol you used in the Hermosura paper, then we haven't tried that. However, something we have been looking at recently is that, if we activate a large Ca2+ signal in an intact cell with a supramaximal agonist concentration and then look at ARC currents, they are much smaller (consistent with the calcineurin-dependent inhibition story).
2. I see no problem for both PLCbeta and PLCgamma converging on the same SOC. They will both result in the generation of IP3 which, if produced in sufficient quantity, will deplete the stores and activate SOC.
As for the second part of your question, this obviously relates, at least in part, to the first question re. how ubiquitous we think the ARC channel pathway is.
In the HEK cell case, the agonist-induced generation of AA seems to be exclusively via a cPLA2 that is activated simultaneously, and in parallel, to the PLC pathway. However, it is clear that different cells have several different ways of making AA in response to agonist action – some are inndependent of PLC, some are downstream of PLC. Dissecting all these possibilities out pharmacologically would likely be difficult.
I think the key point to remember, as I stated in a comment I made in response to a posting by Mike Berridge earlier in this discussion, is that although we all routinely talk about PLC-coupled receptors, we should always be aware that this does not necessarily represent the sole signaling pathway activated by such receptors.
3. My understanding of the inhibition of CRAC by Ca2+ entry such that it cannot be detected in an unbuffered situation is that this is largely (although possibly not entirely) due to refilling of the stores by the avid SERCA pumps. Isn't this essentially what the data from Anant Parekh's lab says? If we hold the IP3 receptors open (e.g. with adenophostin) we can measure robust SOC (CRAC-like) currents in HEK cells with the cytosol buffered at 400 nM (see JBC 276: 35676), and I think Anant had showed the same effect at even higher concentrations (J. Physiol. 522: 247).
As for ARC limiting CRAC I think the key here is that during agonist activation, the Ca2+ entry through ARC does not, itself, raise global cytosolic Ca2+ - it only helps to induce the repetitive transient (and probably partial) IP3-induced release of Ca2+ from stores. Only when sufficient IP3 is generated to produce a sustained depletion of the stores is SOC activated. The resulting sustained elevation in cytosolic Ca2+ then turns off the ARC channels and Ca2+ entry is entirely via the SOC/CRAC channels – as is demonstrated by its independence from AA generation.
I am sorry these are all rather long answers to short questions, but I hope they help clarify our view of what is happening.