Activation of Store-operated Calcium Channels

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.

Constitutive recycling of the store-operated Ca2+channel Orai1 and its internalization during meiosis

“…A potential role for vesicular transport in SOCE was also implied through the effect of interventions that altered the cortical cytoskeleton (Patterson et al, 1999;Rosado et al, 2000). More specific approaches targeted at inhibiting different components of the SNARE machinery required for vesicular fusion also produced conflicting results (Yao et al, 1999;Alderton et al, 2000;Bakowski et al, 2003;Scott et al, 2003). It is difficult to reconcile these early findings with our current knowledge of the SOCE activation mechanism centered around STIM1-Orai1 modulation and interaction.…”

SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles

“…In addition, the immunoblot analysis unequivocally showed that SNAP25 as well as SNAP23 is expressed in HeLa cells ( ), although previous studies reported that SNAP25 was expressed predominantly in neuronal tissues while SNAP23 was expressed ubiquitously ( Ravichandran et al, 1996 ; Scott et al, 2003 ).…”

MT1-MMP recruits the ER-Golgi SNARE Bet1 for efficient MT1-MMP transport to the plasma membrane

“…The dominant-negative mutants of transmembrane domain (TMD)-containing SNAREs were constructed by deleting the TMD by PCR-based methods. The mutants of TMDless SNAREs (STX11, SNAP23, SNAP29, and Ykt6) were generated according to previous studies (Scott et al, 2003;Fukasawa et al, 2004;Offenhäuser et al, 2011). For BiFC, Vn (aa 1-172) and Vc (aa 154-236) fragments of Venus were fused with the N-terminus of Bet1 and the C-terminus of MT1-MMP, respectively, in the pCI vector (Promega).…”