A Novel Mechanism For The Store-Operated Calcium Influx Pathway

LETTERS

A novel mechanism for the store-operated calcium influx pathway
Tarik Smani1,3, Sergey I. Zakharov1,3, Peter Csutora2, Endri Leno1, Elena S. Trepakova1 and Victoria M. Bolotina1,4
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 remainsunclear. Recently, we demonstrated1 that native SOC channels2 can be activated by calcium influx factor (CIF)3 that is produced upon depletion of calcium stores4,5, and showed that Ca2+-independent phospholipase A2 (iPLA2) has an important role in the storeoperated calcium influx pathway6. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We alsopropose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA2, resulting in activation of iPLA2 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 iPLA2, inhibits it, and the activity of SOC channels and capacitative calcium influxis terminated. Activation of specific calcium-conducting channels in the plasma membrane is triggered by depletion of intracellular calcium stores in a wide variety of cell types, but the exact molecular mechanism of such communication remains controversial7–9. Recently, new evidence was obtained supporting the hypothesis that such communication could be mediated by CIF3, which is produced by theendoplasmic reticulum after depletion of calcium stores5,10. We demonstrated that CIF can activate1 native store-operated cation channels2 in excised membrane patches, but it remained unclear if CIF activates the channels directly, or whether some additional membrane-delimited signalling cascade is involved. Previously, iPLA2 has not been thought to be an important component of the store-operatedpathway, but several intriguing features of iPLA2 (for review see ref. 11) suggest its potential involvement. Indeed, it is calcium-independent and works in the presence of strong calcium chelators, but it is also regulated by CaM12,13. In addition, iPLA2 is activated after depletion of calcium stores14, although it is unclear how and why. Recently we obtained experimental evidence6 thatexpression and functional activity of iPLA2 may be crucial for activation of capacitative calcium influx mediated by SOCs, such as calcium-selective SOC (Ca2+-SOC; also known as CRAC15) and nonselective cation SOC (cat-SOC2), but the molecular mechanism of iPLA2-dependent signal transduction remains unclear. Here we identify a cascade of previously unexpected plasmamembrane-delimited reactions and proposea novel model for activation of SOC channels. We demonstrate that: first, displacement of inhibitory CaM from iPLA2 mimics activation of SOC and capacitative calcium influx; second, CIF displaces CaM from iPLA2 and causes its activation, which is identical to the activation produced by depletion of calcium stores; third, lysophospholipid products of iPLA2 activate SOC in excised membrane patchesand capacitative calcium influx in intact cells. First we demonstrate that iPLA2 is localized predominantly to the plasma membrane (Fig.1a), suggesting its possible involvement in plasma-membrane-delimited processes. To explore how iPLA2 could be involved in activation of SOC channels, we used smooth muscle cells (SMCs) as a model in which iPLA2 and capacitative calcium influx are present,non-selective cation SOC could be resolved at the single channel level2, and membrane-delimited interaction of SOC with iPLA2 could be studied in excised membrane patches. One of the important features of iPLA2 is that it exists in a complex with CaM, which keeps it in a catalytically inactive state; removal of CaM results in iPLA2 activation12. Molecular and structural studies showed13,16 that in the...