Under physiological conditions of weak intracellular Ca2+ buffering (0.1mM EGTA), the second messenger Ins(1,4,5)P3 often fails to activate any detectable store-operated Ca2+ current. However, it has been reported that the fungal metabolite adenophostin A [which has a severalfold higher affinity than Ins(1,4,5)P3 for Ins(1,4,5)P3 receptors] consistently activates the current under similar conditions. Here, whole-cell patch clamp experiments have been performed to examine how adenophostin A can activate the store-operated Ca2+ current (ICRAC) in RBL-1 (rat basophilic leukaemia) cells. In a strong intracellular Ca2+ buffer, saturating concentrations of adenophostin A activated ICRAC maximally and the current amplitude and kinetics were indistinguishable from those obtained with high concentrations of Ins(1,4,5)P3. In a weak Ca2+ buffer, adenophostin A consistently activated ICRAC, but the current was submaximal. High concentrations of Ins(1,4,5)P3 or the non-metabolizable analogue Ins(2,4,5)P3 were largely ineffective under these conditions. The size of ICRAC to adenophostin A in weak Ca2+ buffer could be significantly increased by either inhibiting sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (‘SERCA’) pumps with thapsi-gargin or enhancing mitochondrial Ca2+ uptake, although blocking the mitochondrial Ca2+ uniporter with Ruthenium Red did not suppress the activation of the current. Changing the levels of free ATP in the recording pipette did not enhance the size of ICRAC evoked by adenophostin A. We also examined two structurally distinct analogues of adenophostin A (manno-adenophostin and ribophostin), for which the affinities for the Ins(1,4,5)P3 receptor are similar to that of Ins(1,4,5)P3 in equilibrium binding experiments. Although these analogues were able to activate ICRAC to its maximal extent in strong buffer, ribophostin, but not manno-adenophostin, consistently activated the current in weak buffer. We conclude that adenophostin A and ribophostin are able to activate ICRAC in weak buffer through a mechanism that is quite distinct from that employed by Ins(1,4,5)P3 and manno-adenophostin and is not related to equilibrium affinities.

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