Say you have a calcium ionophore, which would normally transport Ca2+ across the plasma membrane to increase cytosolic [Ca2+]. Now suppose you have a Ca2+ free buffer but still treated with a calcium ionophore. Would the ionophore cross the plasma membrane and then cross the ER membrane to bind Ca2+ and then transport it out of the ER to increase the cytosol [Ca2+]? Or would it have no effect?
Morgan and Jacob reported that ionomycin enhances Ca2+ influx by stimulationg store-regulated cation entry but not by a direct action at the plasma membrane (Biochemical J, 1994). In that paper, they observed that ionomycin-treatemnt induced the transiently intracellular Ca2+ response under nominally Ca2+-free medium.
I recall a Letter in Nature way back in 80's that A23187 (as it was known before it started to be called Ca ionophore) is a chaeotropic agent. A calcium free buffer alone would not guarantee lack of Ca after you add your biological sample to it. The effect needs to be titrated against EGTA with the caveat that it is not the direct effect of a tetravalent anion, which was also shown in a couple of papers in BBA.
We used A23187 on red blood cells suspended in the medium which was nominally Ca2-free and supplemented with 0.1 mM EGTA. We clearly saw Ca2+ depletion in the cytosol after several hours of storage. So, I believe, after some time you will see a reduction in Ca2+ filling of the stored.
As ionophore it will facilitate the "flow" of calcium over membranes according to the electrochemical potential. As the intracellular stores have a high calcium concentrations, ionomycin will transport calcium from the store into the cytosol.
The ionophore A23187 will distribute itself in all membranes, plasma membrane and in the mebranes of all intracellular organelles with membrane/medium partition ratio > 60. It will mediate the exchanges of any divalent cations available on each side of each membrane towards equilibria given by
[M2+]i/[M2+]o = ([H+]o/[H+]i)^2
where i and o represent membrane sides. In Ca2+-free media, it will tend to deplete internal Ca2+ and mediate Mg transport mainly.
Experimental evidence for the above description may be found in Tiffert et al. (2000). Functional state of the plasma membrane Ca2+ pump in Plasmodium falciparum infected red cells. J. Physiol., 525.1, 125-134, and in Simonsen at al., (1982). Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells. Biochim. Biophys. Acta 692, 431 440.
It depends on the lipophilicity of the unbound ionophore. I cannot find details for the Ca2+ ionophores but valinomycin is much less lipophilic in its unbound form than its K+ bound form. If this is true for Ca2+ ionophores, you may need to use more to remove the Ca2+. You should be able to see a rise in cytosolic Ca2+ if the Ca2+ ionophores are successfully shuttling Ca2+ from organelles into the cytosol.
As Dr. Lew indicated, A23187 is a Calcium/proton exchanger. It will cause calcium ion release from the ER and acidify the ER.
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