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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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A representative Orai1-YFP image and its corresponding mask.TIRF image was acquired as described in Figure 6, and Orai1-YFP cluster boundaries were automatically detected using a localized thresholding method in Matlab. Shown here is a representative image and its corresponding binary cluster mask.DOI:http://dx.doi.org/10.7554/eLife.00802.017
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fig6s1: A representative Orai1-YFP image and its corresponding mask.TIRF image was acquired as described in Figure 6, and Orai1-YFP cluster boundaries were automatically detected using a localized thresholding method in Matlab. Shown here is a representative image and its corresponding binary cluster mask.DOI:http://dx.doi.org/10.7554/eLife.00802.017

Mentions: Because α-SNAP binds the CAD domain of Stim1, we wondered whether α-SNAP contributes to the formation of store-depletion induced Stim1 clusters in the junctional ER. To observe these plasma membrane proximal regions of ER in live cells and to determine whether α-SNAP depletion affects Stim1 clustering in the junctional ER, we used Total Internal Reflection Fluorescence (TIRF) Microscopy. While the ability of Stim1 to oligomerize and translocate to the junctional ER upon store-depletion appeared normal in α-SNAP deficient cells, the size of Stim1-Orai1 clusters appeared larger (Figure 6A). We adopted a localized thresholding method to detect Stim1-Orai1 cluster boundaries (Figure 6—figure supplement 1), used it to quantify the size and intensity of individual Stim1–Orai1 clusters, and found that α-SNAP depletion increased the overall size of CRAC channel clusters (Figure 6B). Because Orai1 is thought to be largely dependent on Stim1 for its clustering in ER–PM junctions (Liou et al., 2005; Zhang et al., 2005; Stathopulos et al., 2006; Wu et al., 2006; Liou et al., 2007; Varnai et al., 2007; Luik et al., 2008; Stathopulos et al., 2008), we hypothesized that this increase in cluster size could result from an increase in the density of Stim1 in the junctional ER or could reflect a specific defect in the ability of Stim1 to efficiently co-cluster Orai1. To distinguish between these possibilities, we first quantified the average intensity of CFP-Stim1 in individual Stim1–Orai1 clusters. We did not find an appreciable difference in the average intensity of CFP-Stim1 estimated from Stim1–Orai1 clusters in control or α-SNAP depleted, HEK 293 cells stably expressing CFP-Stim1 and Orai1-YFP (Figure 6—figure supplement 2). These data suggest that α-SNAP depletion does not affect the density of Stim1 in junctional clusters. Surprisingly, we found a significant increase in the average Orai1-YFP intensity in α-SNAP depleted clusters (Figure 6—figure supplement 2), resulting in a significant decrease in the ratio of CFP (Stim1) to YFP (Orai1) across all CRAC channel clusters (Figure 6C) as well as in the majority of individual clusters (Figure 6D). These changes and their inhibitory effect on SOCE (Figure 6—figure supplement 3) suggest that α-SNAP regulates an active molecular rearrangement within CRAC channel clusters that is necessary for obtaining optimal Stim1/Orai1 ratios required for the physiological activation of SOCE through CRAC channels. Indeed, two independent recent studies that experimentally manipulated Stim1:Orai1 ratios in junctional clusters showed that the amplitude of calcium selective CRAC currents correlates well with the ratio of Stim1 to Orai1 or CAD to Orai1 within CRAC channel complex (Mcnally et al., 2012; Hoover and Lewis, 2011).10.7554/eLife.00802.016Figure 6.α-SNAP regulates the molecular composition of CRAC channel clusters in ER–PM junctions.


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)

A representative Orai1-YFP image and its corresponding mask.TIRF image was acquired as described in Figure 6, and Orai1-YFP cluster boundaries were automatically detected using a localized thresholding method in Matlab. Shown here is a representative image and its corresponding binary cluster mask.DOI:http://dx.doi.org/10.7554/eLife.00802.017
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6s1: A representative Orai1-YFP image and its corresponding mask.TIRF image was acquired as described in Figure 6, and Orai1-YFP cluster boundaries were automatically detected using a localized thresholding method in Matlab. Shown here is a representative image and its corresponding binary cluster mask.DOI:http://dx.doi.org/10.7554/eLife.00802.017
Mentions: Because α-SNAP binds the CAD domain of Stim1, we wondered whether α-SNAP contributes to the formation of store-depletion induced Stim1 clusters in the junctional ER. To observe these plasma membrane proximal regions of ER in live cells and to determine whether α-SNAP depletion affects Stim1 clustering in the junctional ER, we used Total Internal Reflection Fluorescence (TIRF) Microscopy. While the ability of Stim1 to oligomerize and translocate to the junctional ER upon store-depletion appeared normal in α-SNAP deficient cells, the size of Stim1-Orai1 clusters appeared larger (Figure 6A). We adopted a localized thresholding method to detect Stim1-Orai1 cluster boundaries (Figure 6—figure supplement 1), used it to quantify the size and intensity of individual Stim1–Orai1 clusters, and found that α-SNAP depletion increased the overall size of CRAC channel clusters (Figure 6B). Because Orai1 is thought to be largely dependent on Stim1 for its clustering in ER–PM junctions (Liou et al., 2005; Zhang et al., 2005; Stathopulos et al., 2006; Wu et al., 2006; Liou et al., 2007; Varnai et al., 2007; Luik et al., 2008; Stathopulos et al., 2008), we hypothesized that this increase in cluster size could result from an increase in the density of Stim1 in the junctional ER or could reflect a specific defect in the ability of Stim1 to efficiently co-cluster Orai1. To distinguish between these possibilities, we first quantified the average intensity of CFP-Stim1 in individual Stim1–Orai1 clusters. We did not find an appreciable difference in the average intensity of CFP-Stim1 estimated from Stim1–Orai1 clusters in control or α-SNAP depleted, HEK 293 cells stably expressing CFP-Stim1 and Orai1-YFP (Figure 6—figure supplement 2). These data suggest that α-SNAP depletion does not affect the density of Stim1 in junctional clusters. Surprisingly, we found a significant increase in the average Orai1-YFP intensity in α-SNAP depleted clusters (Figure 6—figure supplement 2), resulting in a significant decrease in the ratio of CFP (Stim1) to YFP (Orai1) across all CRAC channel clusters (Figure 6C) as well as in the majority of individual clusters (Figure 6D). These changes and their inhibitory effect on SOCE (Figure 6—figure supplement 3) suggest that α-SNAP regulates an active molecular rearrangement within CRAC channel clusters that is necessary for obtaining optimal Stim1/Orai1 ratios required for the physiological activation of SOCE through CRAC channels. Indeed, two independent recent studies that experimentally manipulated Stim1:Orai1 ratios in junctional clusters showed that the amplitude of calcium selective CRAC currents correlates well with the ratio of Stim1 to Orai1 or CAD to Orai1 within CRAC channel complex (Mcnally et al., 2012; Hoover and Lewis, 2011).10.7554/eLife.00802.016Figure 6.α-SNAP regulates the molecular composition of CRAC channel clusters in ER–PM junctions.

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