title: Activation of store-operated calcium channels: assessment of the role of snare-mediated vesicular transport.
abstract: Store-operated calcium channels (SOC) play a central role in cellular calcium homeostasis. Although it is well established that SOC are activated by depletion of the endoplasmic reticulum calcium stores, the molecular mechanism underlying this effect remains ill defined. It has been suggested that SOC activation requires fusion of endomembrane vesicles with the plasmalemma. In this model, SNARE-dependent exocytosis is proposed to deliver channels or their activators to the surface membrane to initiate calcium influx. To test this hypothesis, we studied the requirement for membrane fusion events in SOC activation, using a variety of dominant-negative constructs and toxins that interfere with SNARE function. Botulinum neurotoxin A (BotA), which cleaves SNAP-25, did not prevent SOC activation. Moreover, SNAP-25 was not detectable in the cells where BotA was reported earlier to inhibit SOC. Instead, the BotA-insensitive SNAP-23 was present. Impairment of VAMP function was similarly without effect on SOC opening. We also tested the role of N-ethylmaleimide-sensitive factor, a global regulator of SNARE-mediated membrane fusion. Expression of a mutated N-ethylmaleimide-sensitive factor construct inhibited all aspects of membrane traffic tested, including recycling of transferrin receptors to the plasma membrane, fusion of endosomes with lysosomes, and retrograde traffic to the Golgi complex. Despite this global inhibition of vesicular fusion, which was accompanied by gross alterations in cell morphology, SOC activation persisted. These observations cannot be easily reconciled with the vesicle-mediated coupling hypothesis of SOC activation. Our findings imply that the SOC and the machinery necessary to activate them exist in the plasma membrane or are associated with it prior to activation.
authors.affiliation: Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
authors.forename: Cameron C
authors.initials: CC
authors.lastname: Scott
authors.forename: Wendy
authors.initials: W
authors.lastname: Furuya
authors.forename: William S
authors.initials: WS
authors.lastname: Trimble
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000494:pharmacology; D014162:Transfection; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins
chemical_list: D015220:Calcium Channels; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; C494208:SNAP25 protein, human; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D002118:Calcium