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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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Quantification of Stim1-Orai1 cluster size in α-SNAP over-expressing cells.CFP-Stim1 and Orai1-YFP expressing stable HEK 293 cells co-expressing α-SNAP (red) or empty vector control (black) from Figure 6E and 6F were quantified as in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.00802.022
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fig6s6: Quantification of Stim1-Orai1 cluster size in α-SNAP over-expressing cells.CFP-Stim1 and Orai1-YFP expressing stable HEK 293 cells co-expressing α-SNAP (red) or empty vector control (black) from Figure 6E and 6F were quantified as in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.00802.022

Mentions: A fraction of α-SNAP binds Stim1 constitutively, however, binding to Orai1 likely stabilizes its interaction with and enhances its recruitment to the CRAC channel clusters since co-expression of the three proteins shows a significant increase in the percentage of cells showing co-localization of α-SNAP with CRAC channel clusters. In turn, co-expression of α-SNAP synergizes with Stim1 and Orai1 to amplify SOCE likely by ensuring that a higher percentage of Stim1–Orai1 clusters form functional CRAC channels. Interestingly, like in the case of α-SNAP depleted cells, we observed an increase in the size of CRAC channel clusters even in cells over-expressing α-SNAP (Figure 6—figure supplement 6), suggesting that the ratio of Stim1:Orai1 molecules within CRAC channel clusters determines the amplitude of SOCE rather than the size of clusters themselves or the absolute amount of Stim1 in the junctional ER. The increase in size could result from a disrupted stoichiometry between α-SNAP and a fraction of Stim1–Orai1 clusters in α-SNAP depleted as well as over-expressing cells. Alternatively, expansion in cluster size could result from an increase in the total amount of Stim1 in the junctional ER (Wu et al., 2006). Future studies employing sub-diffraction, single molecule imaging approaches would help elucidate the dynamics of α-SNAP dependent re-arrangement of Stim1-Orai1 molecules within junctional CRAC channel clusters.


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)

Quantification of Stim1-Orai1 cluster size in α-SNAP over-expressing cells.CFP-Stim1 and Orai1-YFP expressing stable HEK 293 cells co-expressing α-SNAP (red) or empty vector control (black) from Figure 6E and 6F were quantified as in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.00802.022
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6s6: Quantification of Stim1-Orai1 cluster size in α-SNAP over-expressing cells.CFP-Stim1 and Orai1-YFP expressing stable HEK 293 cells co-expressing α-SNAP (red) or empty vector control (black) from Figure 6E and 6F were quantified as in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.00802.022
Mentions: A fraction of α-SNAP binds Stim1 constitutively, however, binding to Orai1 likely stabilizes its interaction with and enhances its recruitment to the CRAC channel clusters since co-expression of the three proteins shows a significant increase in the percentage of cells showing co-localization of α-SNAP with CRAC channel clusters. In turn, co-expression of α-SNAP synergizes with Stim1 and Orai1 to amplify SOCE likely by ensuring that a higher percentage of Stim1–Orai1 clusters form functional CRAC channels. Interestingly, like in the case of α-SNAP depleted cells, we observed an increase in the size of CRAC channel clusters even in cells over-expressing α-SNAP (Figure 6—figure supplement 6), suggesting that the ratio of Stim1:Orai1 molecules within CRAC channel clusters determines the amplitude of SOCE rather than the size of clusters themselves or the absolute amount of Stim1 in the junctional ER. The increase in size could result from a disrupted stoichiometry between α-SNAP and a fraction of Stim1–Orai1 clusters in α-SNAP depleted as well as over-expressing cells. Alternatively, expansion in cluster size could result from an increase in the total amount of Stim1 in the junctional ER (Wu et al., 2006). Future studies employing sub-diffraction, single molecule imaging approaches would help elucidate the dynamics of α-SNAP dependent re-arrangement of Stim1-Orai1 molecules within junctional CRAC channel clusters.

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