Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.

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Depletion of intracellular Ca2؉ stores activates capacitative Ca 2؉ influx in smooth muscle cells, but the native storeoperated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca 2؉ stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of storeoperated channels, which are activated upon passive depletion of Ca 2؉ stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca 2؉ ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Load-ing SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid), which slowly depletes Ca 2؉ stores without a rise in intracellular Ca (4, 8) and certain members of the diverse family of TRP channels (9, 10) are thought to be responsible for CCE. However, to date the existence of neither of those has been established in SMC. Importantly, depletion of Ca 2ϩ stores was shown to trigger not only Ca 2ϩ , but also Na ϩ influx in arterial myocytes (11), which implies that store-operated channels in SMC are poorly selective for cations. In freshly isolated mouse anococcygeus SMC there are strong indications that CCE results from activation of a whole cell nonselective cation current (12, 13), although in rat aortic SMC line A7r5 no currents were detected which could be associated with CCE (14, 15). It is totally unclear if the same or different storeoperated channels mediate CCE in SMC from different preparations.Here for the first time we characterize 3-pS cation channels that are activated by Ca 2ϩ store depletion in intact SMC from mouse and rabbit aorta. These channels, contrary to highly Ca 2ϩ -selective CRAC channels, are poorly selective for monoand divalent cations, and under physiological conditions they will allow both Ca 2ϩ and Na ϩ to enter SMC. Recently we found that these channels can also be activated in excised membrane patches by calcium influx factor (CIF) partially purified from human platelets or yeast with depleted Ca 2ϩ stores (16). Taken together, these data strongly support the idea that the native 3-pS channels, which we found in SMC, belong to the class of store-operated ion channels. Preliminary data have been reported in abstract form (17, 18). EXPERIMENTAL PROCEDURES SMC PreparationFour different preparations of aortic SMC were used in our experiments, and the 3-pS channel described in this paper was found to be the same in acutely dispersed and cultured SMC from mouse and rabbit aorta. Most of the experiments on characterization of the single channels and whole cell currents were done on mSMC in short term culture because they pr...

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Abstract-Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca 2ϩ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca 2ϩ /Mn 2ϩ influx in intact platelets and to monitor Ca 2ϩ uptake into the stores in permeabilized platelets, fura-2 was used. 4 -6 Similar to other types of nonexcitable cells, 7 agonist-activated Ca 2ϩ influx in platelets is thought to be capacitative in nature, being also activated by passive store depletion with an inhibitor(s) of sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA), thapsigargin (TG) or 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). 8 -11 Nitric oxide (NO), a potent inhibitor of thrombin-activated platelet aggregation, 12,13 is known to decrease Ca 2ϩ cyt that is raised by agonists, 14 although the mechanism is not well defined. It has been shown that NO inhibits thrombin-and IP 3 -stimulated Ca 2ϩ release from internal stores in intact 15 and permeabilized platelets. 16,17 However, it is still unclear whether NO also inhibits capacitative cation influx. Brune et al 17 found a significant inhibitory effect of the NO donor, sodium nitroprusside, on Mn 2ϩ influx in TG-treated platelets. In contrast, Okamoto et al 18 did not find an effect of the NO donor on TG-induced Ca 2ϩ influx in human platelets and concluded that capacitative Ca 2ϩ influx is resistant to NO. The data presented here demonstrate that NO, indeed, is a potent inhibitor of capacitative cation influx in human platelets and that SERCA activity is required for this effect. This finding is consistent with the idea that NO inhibits capacitative cation influx indirectly by promoting SERCA-dependent refilling of intracellular Ca 2ϩ stores. Materials and Methods Platelet Isolation ProcedureBlood from healthy adult volunteers was drawn into plastic nonsterile tubes containing 10% volume of anticoagulant. The composition of anticoagulant was (in g/100 mL): trisodium citrate 2.5, citric acid