E-Conference: Defining Calcium Entry Signals
How many stores are there?
12 June 2004
Thanks for the thoughtful reply. I will try and answer some of the questions you raised and comment on some of the points you made.
"1. This point about the action of very low subthreshold doses is well taken. I very much like your observation that CRAC can be activated at 30nM carbachol without inducing release, has this been published?"
Yes, but it's in a rather obscure journal [Hermosura et al. (2000) Nature 408,735-740] ;-)
"An essential component of my hypothesis is that entry occurs before release and I suggested a possible mechanism for this in my hypothesis. I suggested that the formation of IP3 was highly localized in the immediate vicinity of the receptor, exactly as you do, where it could have two possible actions. Either it could activate conformation coupling directly or it could induce a local release to switch on entry through local store emptying."
Yes, I completely agree that local signaling under the plasma membrane is crucial at low agonist concentrations. I'm not a big fan of the direct coupling hypothesis via IP3R, since SOC works just fine without IP3R (in DT40 cells with triple IP3R KO) and huge doses of heparin completely block IP3-induced activation of CRAC without affecting CRAC that is activated by e.g. ionomycin (I know, you can always argue that the coupling is independent of IP3, but I would expect heparin to at least hinder the interaction of IP3R with other proteins).
"2. (...) You seem to be concerned about why there is no plateau phase during this threshold condition. As I argued before, the reason why there is no plateau phase is because the SERCA pumps are very efficient at taking up the Ca2+ and it this uptake that sensitizes the IP3Rs."
I'm not really concerned about the lack of the plateau at threshold agonist concentrations, because I do have an explanation for it, although it's different from the one you propose. If I understand you correctly (please correct me if I'm wrong), you seem to assume that influx always precedes release and that the influx supercharges the bulk ER so it becomes more sensitive to IP3. Our data would argue against that. As mentioned in my previous post, the bulk ER responds very sensitively to IP3. 1 micromolar IP3 administered via patch pipette (i.e. globally) will essentially empty the entire IP3-sensitive store, but that is not enough to trigger CRAC [see Parekh et al. (1997) Cell 89, 973-980]. So the bulk of the ER is clearly more sensitive to IP3 than the CRAC store (as to whether this is functionally or physically separate is not important in this context, although I would argue that this experiment is more consistent with physical separation). In any case, a reasonable conclusion from this is that for whatever reasons, the CRAC store has a higher threshold for IP3 than the bulk of the ER. Yet, when we go to subthreshold agonist concentrations it can be emptied first and cause influx. Based on a number observations, we have proposed that this is due to local metabolism of IP3 in the vicinity of the CRAC stores [see Hermosura et al. (2000) Nature 408, 735-740]. So, let me briefly compare the subtreshold and the threshold scenario:
Subthreshold: CRAC stores are selectively emptied due to local inhibition of IP3 metabolizing enzymes. This only works when subthreshold agonist doses are delivered repeatedly. The first application does not trigger anything (neither influx nor release; subthreshold). However, it primes the system by generating IP4, which is a very potent inhibitor of IP3 5-phosphatase and more long-lived than IP3 itself. Deeper ER is not affected, because IP3 remains local and subthreshold. Now, when the second subthreshold agonist application occurs, the IP3 metabolism underneath the plasma membrane is compromised and this causes an apparent sensitization towards IP3. This now is above the threshold to trigger the CRAC stores, resulting in only Ca2+ influx, but no release transient, because the IP3 increase remains local.
Threshold: Here, the higher IP3 concentrations escape the subplasma membrane domain to trigger the more sensitive bulk ER release, and we get a Ca2+ transient. It is possible that the higher IP3 concentrations also overwhelm the metabolizing enzymes around the CRAC store and trigger the CRAC stores. However, the significant Ca2+ release from the bulk ER may cause a rapid shutdown of the slowly activating CRAC due to the uptake of Ca2+ into the CRAC store (but see my comment at the very end of this post). That is my favorite explanation for the lack of a plateau.
"Perhaps the main difference in our interpretation is whether there is a single or two separate stores (a CRAC store and a larger release store). I dealt with this question in my comments. It seems that we both agree that there has to be two functional stores and the question therefore is whether or not they are physically separate. I argue that the ER is continuous but that it can be separated physiologically into two regions. The junctional zone that responds to the high dose of IP3 near the membrane and the remainder of the store where the uncoupled IP3Rs are located. At physiological agonist concentrations, these uncoupled receptors do not respond directly to IP3 because the concentration is too low. Therefore, before they can respond to the low ambient IP3 level they have to be sensitized by the Ca2+ coming in from the outside through the entry pathway."
I guess we have different ways of explaining the same phenomenon. You say that the bulk ER is initially less sensitive to IP3 than the CRAC store and requires Ca2+ entry to increase its sensitivity, whereas I argue the opposite, namely that the bulk ER is more sensitive than the CRAC store, but the CRAC store can gain apparent sensitivity when IP3 metabolism is altered. I would argue that the IP3 dose response curve obtained with patch pipette-loaded IP3 appears to support a higher sensitivity of the bulk ER. Adding IP4 changes the CRAC store sensitivity in these very same conditions.
"As I mentioned in my comments, the problem with having two separate stores is what happens when the small CRAC store fills up and the main release pool is still empty?"
Another question is whether the bulk ER ever gets emptied while the CRAC store is full. If so, then we need to ask the question whether filling the CRAC actually turns off CRAC. Really, we have no clue what activates CRAC and that represents a wealth of knowledge compared to what we know about the mechansims that would turn it off :-)
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