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An essential and NSF independent role for α-SNAP in store-operated calcium entry.

Miao Y, Miner C, Zhang L, Hanson PI, Dani A, Vig M - Elife (2013)

Bottom Line: Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined.Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE.This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly.

View Article: PubMed Central - PubMed

Affiliation: Pathology and Immunology , Washington University School of Medicine , St Louis , United States.

ABSTRACT
Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca(2+) sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE. DOI:http://dx.doi.org/10.7554/eLife.00802.001.

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Morphology of ER and Golgi in control and α-SNAP depleted cells.Epifluorescence images of α-SNAP or scr RNAi treated HEK 293 cells, stained with anti-calreticulin (Left Panel), or anti-giantin (Right Panel) primary antibodies followed by respective secondary antibodies. Scale bar 10 μm. (n = 3)DOI:http://dx.doi.org/10.7554/eLife.00802.013
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fig3s3: Morphology of ER and Golgi in control and α-SNAP depleted cells.Epifluorescence images of α-SNAP or scr RNAi treated HEK 293 cells, stained with anti-calreticulin (Left Panel), or anti-giantin (Right Panel) primary antibodies followed by respective secondary antibodies. Scale bar 10 μm. (n = 3)DOI:http://dx.doi.org/10.7554/eLife.00802.013

Mentions: α-SNAP is well known for its ability to promote SNARE complex disassembly and SNARE protein recycling by bridging NSF to SNARE complexes (Clary et al., 1990; Hanson et al., 1995; Jahn et al., 1995; Xu et al., 1999; Marz et al., 2003; Barszczewski et al., 2008; Wickner and Schekman, 2008; Winter et al., 2009; Chang et al., 2012). Therefore, α-SNAP depletion could affect Orai1 trafficking to the PM or Stim1 localization in the ER, thereby contributing to defective SOCE. Epifluorescence imaging of Orai1-YFP and YFP-Stim1 expressing α-SNAP depleted cells showed normal localization of both Orai1 and Stim1 (Figure 3A and 3B). To specifically quantify Orai1 protein levels at the PM, we tagged the extracellular loop of Orai1 with a α-bungarotoxin (BTX) binding site (BBS) (Sekine-Aizawa and Huganir, 2004) (Figure 3—figure supplement 1). Stable expression of Orai1-BBS-YFP followed by exogenous application of labeled BTX provided an estimate of cell surface Orai1 levels. α-SNAP depleted, resting, or store depleted cells did not show any significant difference in the levels of cell surface Orai1 when compared to controls (Figure 3—figure supplement 2). Consistently, immunostaining of α-SNAP depleted cells with an ER marker, calreticulin (Figure 3—figure supplement 3), and a Golgi-marker, giantin (Figure 3—figure supplement 3) showed no apparent changes in the ER-Golgi morphology suggesting that at the time points used in our studies, α-SNAP depletion had not perturbed overall organelle structure.10.7554/eLife.00802.010Figure 3.Regulation of SOCE by α-SNAP is NSF independent.


An essential and NSF independent role for α-SNAP in store-operated calcium entry.

Miao Y, Miner C, Zhang L, Hanson PI, Dani A, Vig M - Elife (2013)

Morphology of ER and Golgi in control and α-SNAP depleted cells.Epifluorescence images of α-SNAP or scr RNAi treated HEK 293 cells, stained with anti-calreticulin (Left Panel), or anti-giantin (Right Panel) primary antibodies followed by respective secondary antibodies. Scale bar 10 μm. (n = 3)DOI:http://dx.doi.org/10.7554/eLife.00802.013
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3713520&req=5

fig3s3: Morphology of ER and Golgi in control and α-SNAP depleted cells.Epifluorescence images of α-SNAP or scr RNAi treated HEK 293 cells, stained with anti-calreticulin (Left Panel), or anti-giantin (Right Panel) primary antibodies followed by respective secondary antibodies. Scale bar 10 μm. (n = 3)DOI:http://dx.doi.org/10.7554/eLife.00802.013
Mentions: α-SNAP is well known for its ability to promote SNARE complex disassembly and SNARE protein recycling by bridging NSF to SNARE complexes (Clary et al., 1990; Hanson et al., 1995; Jahn et al., 1995; Xu et al., 1999; Marz et al., 2003; Barszczewski et al., 2008; Wickner and Schekman, 2008; Winter et al., 2009; Chang et al., 2012). Therefore, α-SNAP depletion could affect Orai1 trafficking to the PM or Stim1 localization in the ER, thereby contributing to defective SOCE. Epifluorescence imaging of Orai1-YFP and YFP-Stim1 expressing α-SNAP depleted cells showed normal localization of both Orai1 and Stim1 (Figure 3A and 3B). To specifically quantify Orai1 protein levels at the PM, we tagged the extracellular loop of Orai1 with a α-bungarotoxin (BTX) binding site (BBS) (Sekine-Aizawa and Huganir, 2004) (Figure 3—figure supplement 1). Stable expression of Orai1-BBS-YFP followed by exogenous application of labeled BTX provided an estimate of cell surface Orai1 levels. α-SNAP depleted, resting, or store depleted cells did not show any significant difference in the levels of cell surface Orai1 when compared to controls (Figure 3—figure supplement 2). Consistently, immunostaining of α-SNAP depleted cells with an ER marker, calreticulin (Figure 3—figure supplement 3), and a Golgi-marker, giantin (Figure 3—figure supplement 3) showed no apparent changes in the ER-Golgi morphology suggesting that at the time points used in our studies, α-SNAP depletion had not perturbed overall organelle structure.10.7554/eLife.00802.010Figure 3.Regulation of SOCE by α-SNAP is NSF independent.

Bottom Line: Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined.Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE.This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly.

View Article: PubMed Central - PubMed

Affiliation: Pathology and Immunology , Washington University School of Medicine , St Louis , United States.

ABSTRACT
Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca(2+) sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE. DOI:http://dx.doi.org/10.7554/eLife.00802.001.

Show MeSH
Related in: MedlinePlus