title: Adenophostin A and ribophostin, but not inositol 1,4,5-trisphosphate or manno-adenophostin, activate the Ca2+ release-activated Ca2+ current, I(CRAC), in weak intracellular Ca2+ buffer.
abstract: Under physiological conditions of weak intracellular Ca(2+) buffering (0.1 mM EGTA), the second messenger Ins(1,4,5)P(3) often fails to activate any detectable store-operated Ca(2+) current. However, it has been reported that the fungal metabolite adenophostin A [which has a severalfold higher affinity than Ins(1,4,5)P(3) for Ins(1,4,5)P(3) 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 Ca(2+) current (I(CRAC)) in RBL-1 (rat basophilic leukaemia) cells. In a strong intracellular Ca(2+) buffer, saturating concentrations of adenophostin A activated I(CRAC) maximally and the current amplitude and kinetics were indistinguishable from those obtained with high concentrations of Ins(1,4,5)P(3). In a weak Ca(2+) buffer, adenophostin A consistently activated I(CRAC), but the current was submaximal. High concentrations of Ins(1,4,5)P(3) or the non-metabolizable analogue Ins(2,4,5)P(3) were largely ineffective under these conditions. The size of I(CRAC) to adenophostin A in weak Ca(2+) buffer could be significantly increased by either inhibiting sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase ('SERCA') pumps with thapsi-gargin or enhancing mitochondrial Ca(2+) uptake, although blocking the mitochondrial Ca(2+) 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 I(CRAC) 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)P(3) receptor are similar to that of Ins(1,4,5)P(3) in equilibrium binding experiments. Although these analogues were able to activate I(CRAC) 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 I(CRAC) in weak buffer through a mechanism that is quite distinct from that employed by Ins(1,4,5)P(3) and manno-adenophostin and is not related to equilibrium affinities.
authors.affiliation: Laboratory of Molecular and Cellular Signalling, Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, U.K. anant.parekh@physiol.ox.ac.uk
authors.forename: Anant B
authors.initials: AB
authors.lastname: Parekh
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Riley
authors.forename: Barry V L
authors.initials: BV
authors.lastname: Potter
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D004187:Disaccharides / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D013403:Sugar Phosphates / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D002021:Buffers; D002120:Calcium Channel Agonists; D015220:Calcium Channels; D004187:Disaccharides; D007295:Inositol Phosphates; D013403:Sugar Phosphates; C446925:manno-adenophostin; C446924:ribophostin; C085029:adenophostin A; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; C062616:inositol 2,4,5-trisphosphate; D000241:Adenosine