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We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.

A major pathway for stimulated Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this store-mediated Ca(2+) entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca(2+) pools, which are differentially modulated by the actin cytoskeleton. Ca(2+) entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca(2+) entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca(2+) entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca(2+) entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM.

Abstract: We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP3 receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton. [Copyright &y& Elsevier]

Store-operated Ca2+ entry (SOCE), a mechanism regulated by the filling state of the intracellular Ca2+ stores, is a major pathway for Ca2+ influx. Hypotheses to explain the communication between the Ca2+ stores and plasma membrane (PM) have considered both the existence of small messenger molecules, such as a Ca2+-influx factor (CIF), and both stable and de novo conformational coupling between proteins in the Ca2+ store and PM. Alternatively, a secretion-like coupling model based on vesicle fusion and channel insertion in the PM has been proposed, which shares some properties with the de novo conformational coupling model, such as the role of the actin cytoskeleton and soluble N-ethylmaleimide (NEM)-sensitive-factor attachment proteins receptor (SNARE) proteins. Here we review recent progress made in the characterization of the de novo conformational coupling and the secretion-like coupling models for SOCE. We pay particular attention into the involvement of SNARE proteins and the actin cytoskeleton in both SOCE models. SNAREs are recognized as proteins involved in exocytosis, participating in vesicle transport, membrane docking, and fusion. As with secretion, a role for the cortical actin network in Ca2+ entry has been demonstrated in a number of cell types. In resting cells, the cytoskeleton may prevent the interaction between the Ca2+ stores and the PM, or preventing fusion of vesicles containing Ca2+ channels with the PM. These are processes in which SNARE proteins might play a crucial role upon cell activation by directing a precise interaction between the membrane of the transported organelle and the PM. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

We recently reported that store-operated Ca2+ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as ‘secretion-like coupling.’ As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. [ABSTRACT FROM AUTHOR]