We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP 3 ) in capacitative calcium entry and calcium releaseactivated calcium current (I crac ) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with U73122 blocked calcium entry and I crac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I crac . The membrane-permeant IP 3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I crac . However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP 3 receptor because the drug was equally effective in wildtype DT40 B-cells and in DT40 B-cells whose genes for all three IP 3 receptors had been disrupted. Intracellular application of another potent IP 3 receptor antagonist, heparin, failed to inhibit activation of I crac . Finally, the inhibition of I crac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP 3 . These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP 3 and IP 3 receptor in this mechanism, at least in these particular cell types.

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Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca 2؉ , Mg 2؉ , and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca 2؉ stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca In a variety of cells, binding of ligands such as neurotransmitters, hormones, or growth factors to receptors on the cell surface generates intracellular calcium signals. Receptor-mediated release of Ca 2ϩ from IP 3 1 -sensitive intracellular stores triggers an influx of Ca 2ϩ into the cell via store-operated channels (SOCs) in the plasma membrane, a process termed "capacitative calcium entry" (1, 2). The resulting increase in intracellular Ca 2ϩ concentration regulates important processes, ranging from cell growth and differentiation to apoptosis and cell death. Molecular candidates for SOCs are the mammalian homologues of the Drosophila TRP (transient receptor potential) protein (3). Expression of the related genes, however, has resulted in varying patterns of response, including constitutive activity, augmentation of capacitative calcium entry, apparent direct activation by IP 3 , and activation by diacylglycerol (4). Therefore, the mechanism by which emptying of intracellular Ca 2ϩ stores activates SOCs remains elusive. Recent findings have favored a "direct coupling" model, whereby IP 3 receptors in the store membranes sense the Ca 2ϩ -filling status of the stores and transfer this information via direct coupling to subunits of SOCs in the plasma membrane (5-8).For the present, the best knowledge of SOCs comes from electrophysiological studies of a specific current associated with SOCs, known as the Ca 2ϩ release-activated calcium current (I crac ) (9). Despite extensive characterization of the macroscopic CRAC currents, the single-channel signature is poorly understood. This is because the channels are presumed to have a minute single-channel conductance, too low to be resolved at the single-channel level (10). During whole cell measurements in Jurkat T-lymphocytes, recently, Kerschbaum and Cahalan (11) were able to detect for the first time single-channel events which they attributed to CRAC channels. Their approach was to measure currents in the complete absence of intra-and extracellular divalent cations. They reasoned that under these conditions CRAC channels, like voltage-gated Ca 2ϩ channels should pass monovalent cations indiscriminately and with enhanced single-channel conductance (12)(13)(14). Ind...