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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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α-SNAP co-expression augments SOCE in Stim1-Orai1 over-expressing cells.Average Fura-2 ratios of Orai1-Stim1 expressing HEK 293 cells transiently transfected with α-SNAP (red) or empty vector as a control (black) and stimulated with TG to measure SOCE (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00802.020
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fig6s4: α-SNAP co-expression augments SOCE in Stim1-Orai1 over-expressing cells.Average Fura-2 ratios of Orai1-Stim1 expressing HEK 293 cells transiently transfected with α-SNAP (red) or empty vector as a control (black) and stimulated with TG to measure SOCE (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00802.020

Mentions: Given this reduction in the CFP (Stim1) to YFP (Orai1) ratio of α-SNAP deficient junctional clusters, we next wondered whether over-expression of α-SNAP would increase the Stim1/Orai1 ratio in clusters and thereby augment SOCE. Remarkably, over-expression of α-SNAP enhanced SOCE beyond what is typically seen in cells stably co-expressing Stim1 and Orai1 (Figure 6—figure supplement 4). Next, we examined the relative average intensities of CFP and YFP in junctional clusters of cells stably expressing CFP-Stim1, Orai1-YFP and transiently transfected with α-SNAP or empty vector. Concomitant with an increase in SOCE, over-expression of α-SNAP significantly enhanced the ratio of CFP (Stim1) to YFP (Orai1) across all junctional clusters (Figure 6E) as well as majority of individual clusters (Figure 6F). Notably, over-expression of α-SNAP did not affect total surface Orai1 levels in resting or store-depleted cells (Figure 6—figure supplement 5) or average CFP-Stim1 intensity per cell (data not shown).


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)

α-SNAP co-expression augments SOCE in Stim1-Orai1 over-expressing cells.Average Fura-2 ratios of Orai1-Stim1 expressing HEK 293 cells transiently transfected with α-SNAP (red) or empty vector as a control (black) and stimulated with TG to measure SOCE (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00802.020
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6s4: α-SNAP co-expression augments SOCE in Stim1-Orai1 over-expressing cells.Average Fura-2 ratios of Orai1-Stim1 expressing HEK 293 cells transiently transfected with α-SNAP (red) or empty vector as a control (black) and stimulated with TG to measure SOCE (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00802.020
Mentions: Given this reduction in the CFP (Stim1) to YFP (Orai1) ratio of α-SNAP deficient junctional clusters, we next wondered whether over-expression of α-SNAP would increase the Stim1/Orai1 ratio in clusters and thereby augment SOCE. Remarkably, over-expression of α-SNAP enhanced SOCE beyond what is typically seen in cells stably co-expressing Stim1 and Orai1 (Figure 6—figure supplement 4). Next, we examined the relative average intensities of CFP and YFP in junctional clusters of cells stably expressing CFP-Stim1, Orai1-YFP and transiently transfected with α-SNAP or empty vector. Concomitant with an increase in SOCE, over-expression of α-SNAP significantly enhanced the ratio of CFP (Stim1) to YFP (Orai1) across all junctional clusters (Figure 6E) as well as majority of individual clusters (Figure 6F). Notably, over-expression of α-SNAP did not affect total surface Orai1 levels in resting or store-depleted cells (Figure 6—figure supplement 5) or average CFP-Stim1 intensity per cell (data not shown).

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