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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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RNAi mediated depletion of γ-SNAP and measurement of SOCE.(Left Panel) Average Fura-2 ratios of HEK 293 cells transduced with scr RNAi or five different RNAi targeting γ-SNAP, and stimulated with TG to measure SOCE (using flexstation). (Right Panel) Semi-quantitative PCR on total RNA to assess the level of γ-SNAP mRNA depletion compared to GAPDH in RNAi treated cells.DOI:http://dx.doi.org/10.7554/eLife.00802.008
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fig1s5: RNAi mediated depletion of γ-SNAP and measurement of SOCE.(Left Panel) Average Fura-2 ratios of HEK 293 cells transduced with scr RNAi or five different RNAi targeting γ-SNAP, and stimulated with TG to measure SOCE (using flexstation). (Right Panel) Semi-quantitative PCR on total RNA to assess the level of γ-SNAP mRNA depletion compared to GAPDH in RNAi treated cells.DOI:http://dx.doi.org/10.7554/eLife.00802.008

Mentions: To test our hypothesis that additional proteins are needed to facilitate SOCE under physiological conditions, we used ∼200 to 500 base pair long double stranded RNA (dsRNA) to deplete the expression of six genes that we previously identified as candidates in a genome-wide RNAi screen (Vig et al., 2006b) in Drosophila Kc cells. We found that knockdown of soluble NSF attachment protein (SNAP) strongly reduces SOCE by day 3 (Figure 1A and Supplementary file 1). Given the almost complete inhibition of SOCE in SNAP deficient Drosophila cells, we hypothesized that SNAP might engage in novel, SOCE specific protein–protein interactions that may or may not involve NSF and SNAREs. α- and β-SNAP are the two mammalian proteins most closely related to Drosophila SNAP (Figure 1—figure supplement 1). α-SNAP is ubiquitously expressed while β-SNAP expression is largely restricted to the brain. We depleted α-SNAP in HEK-293 (Figure 1B), Jurkat T cells (Figure 1C) and U2OS cells (Figure 1—figure supplement 2) using lentivirus-based RNAi constructs (Supplementary file 1) and found SOCE to be strongly inhibited by day three compared to cells treated with control RNAi. By day five, depletion of α-SNAP caused cell rounding in adherent cell lines; we therefore restricted our analysis to adherent cells at early time-points. We confirmed the efficiency of α-SNAP knockdown for each experiment on western blots of whole cell lysates (WCLs) (Figure 1D) and by immunostaining cells with anti-α-SNAP antibody (Figure 1—figure supplement 3). Importantly, when we reconstituted α-SNAP deficient HEK 293 cells with an RNAi resistant version of α-SNAP we found that SOCE was largely restored (Figure 1E). Defective SOCE in α-SNAP deficient HEK 293 cells could also be restored by over-expressing β-SNAP (Figure 1—figure supplement 4). γ-SNAP is a third SNAP protein widely expressed in mammalian tissues but is less similar to Drosophila SNAP and α-SNAP than β-SNAP (Figure 1—figure supplement 1). γ-SNAP depletion failed to inhibit SOCE in HEK 293 cells (Figure 1—figure supplement 5) and γ-SNAP over-expression did not compensate for α-SNAP depletion (Figure 1—figure supplement 4).10.7554/eLife.00802.003Figure 1.α-SNAP depletion inhibits SOCE and NFAT activation.


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)

RNAi mediated depletion of γ-SNAP and measurement of SOCE.(Left Panel) Average Fura-2 ratios of HEK 293 cells transduced with scr RNAi or five different RNAi targeting γ-SNAP, and stimulated with TG to measure SOCE (using flexstation). (Right Panel) Semi-quantitative PCR on total RNA to assess the level of γ-SNAP mRNA depletion compared to GAPDH in RNAi treated cells.DOI:http://dx.doi.org/10.7554/eLife.00802.008
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1s5: RNAi mediated depletion of γ-SNAP and measurement of SOCE.(Left Panel) Average Fura-2 ratios of HEK 293 cells transduced with scr RNAi or five different RNAi targeting γ-SNAP, and stimulated with TG to measure SOCE (using flexstation). (Right Panel) Semi-quantitative PCR on total RNA to assess the level of γ-SNAP mRNA depletion compared to GAPDH in RNAi treated cells.DOI:http://dx.doi.org/10.7554/eLife.00802.008
Mentions: To test our hypothesis that additional proteins are needed to facilitate SOCE under physiological conditions, we used ∼200 to 500 base pair long double stranded RNA (dsRNA) to deplete the expression of six genes that we previously identified as candidates in a genome-wide RNAi screen (Vig et al., 2006b) in Drosophila Kc cells. We found that knockdown of soluble NSF attachment protein (SNAP) strongly reduces SOCE by day 3 (Figure 1A and Supplementary file 1). Given the almost complete inhibition of SOCE in SNAP deficient Drosophila cells, we hypothesized that SNAP might engage in novel, SOCE specific protein–protein interactions that may or may not involve NSF and SNAREs. α- and β-SNAP are the two mammalian proteins most closely related to Drosophila SNAP (Figure 1—figure supplement 1). α-SNAP is ubiquitously expressed while β-SNAP expression is largely restricted to the brain. We depleted α-SNAP in HEK-293 (Figure 1B), Jurkat T cells (Figure 1C) and U2OS cells (Figure 1—figure supplement 2) using lentivirus-based RNAi constructs (Supplementary file 1) and found SOCE to be strongly inhibited by day three compared to cells treated with control RNAi. By day five, depletion of α-SNAP caused cell rounding in adherent cell lines; we therefore restricted our analysis to adherent cells at early time-points. We confirmed the efficiency of α-SNAP knockdown for each experiment on western blots of whole cell lysates (WCLs) (Figure 1D) and by immunostaining cells with anti-α-SNAP antibody (Figure 1—figure supplement 3). Importantly, when we reconstituted α-SNAP deficient HEK 293 cells with an RNAi resistant version of α-SNAP we found that SOCE was largely restored (Figure 1E). Defective SOCE in α-SNAP deficient HEK 293 cells could also be restored by over-expressing β-SNAP (Figure 1—figure supplement 4). γ-SNAP is a third SNAP protein widely expressed in mammalian tissues but is less similar to Drosophila SNAP and α-SNAP than β-SNAP (Figure 1—figure supplement 1). γ-SNAP depletion failed to inhibit SOCE in HEK 293 cells (Figure 1—figure supplement 5) and γ-SNAP over-expression did not compensate for α-SNAP depletion (Figure 1—figure supplement 4).10.7554/eLife.00802.003Figure 1.α-SNAP depletion inhibits SOCE and NFAT activation.

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