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? 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.
2. You make a distinction between subthreshold and threshold on the basis that the latter is the concentration that begins to give a meaningful Ca²⁺ signal in the form of a Ca²⁺ spike, which relates to a point made by Ken Byron. I am comfortable with this distinction because it fits in very nicely with my hypothesis. The two are really part of a continuum. At the subthreshold dose the entry is not large enough to set up the conditions for release to occur for two reasons. Firstly the level of IP₃ reaching the uncoupled receptors is not high enough to trigger them. Secondly, and more importantly, the level of entry is not large enough to increase the lumenal level of Ca²⁺ sufficiently to sensitize the uncoupled IP₃Rs to the low level of IP₃.

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.



3. With regard to high doses, I agree with you completely.

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.

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?