Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
J Cell Sci. 2011 Aug 1;124(Pt 15):2602-10. doi: 10.1242/jcs.084178. Epub 2011 Jul 12.
We have previously shown that PIP5KIβ and PIP5KIγ generate functionally distinct pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] important for antigen-stimulated Ca(2+) entry in mast cells. In the present study, we find that association of the endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, and the store-operated Ca(2+) channel, Orai1, stimulated by thapsigargin-mediated ER store depletion, is enhanced by overexpression of PIP5KIβ and inhibited by overexpression of PIP5KIγ. These different PIP5KI isoforms cause differential enhancement of PtdIns(4,5)P(2) in detergent-resistant membrane (DRM) fractions, which comprise ordered lipid regions, and detergent-solubilized membrane (DSM) fractions, which comprise disordered lipid regions. Consistent with these results, the inositol 5-phosphatase L10-Inp54p, which is targeted to ordered lipids, decreases PtdIns(4,5)P(2) in the DRM fraction and inhibits thapsigargin-stimulated STIM1-Orai1 association and store-operated Ca(2+) entry, whereas the inositol 5-phosphatase S15-Inp54p, which is targeted to disordered lipids, decreases PtdIns(4,5)P(2) in the DSM fraction and enhances STIM1-Orai1 association. Removal of either the STIM1 C-terminal polylysine sequence (amino acids 677-685) or an N-terminal polyarginine sequence in Orai1 (amino acids 28-33) eliminates this differential sensitivity of STIM1-Orai1 association to PtdIns(4,5)P(2) in the distinctive membrane domains. Our results are consistent with a model of PtdIns(4,5)P(2) balance, in which store-depletion-stimulated STIM1-Orai1 association is positively regulated by the ordered lipid pool of PtdIns(4,5)P(2) and negatively regulated by PtdIns(4,5)P(2) in disordered lipid domains.
我们之前已经表明，PIP5KIβ 和 PIP5KIγ 生成功能不同的磷脂酰肌醇 4,5-二磷酸 [PtdIns(4,5)P(2)] 池，对于抗原刺激的肥大细胞中 Ca(2+)内流至关重要。在本研究中，我们发现内质网 (ER) Ca(2+) 传感器 STIM1 与受 thapsigargin 介导的 ER 储存耗竭刺激的储存操作 Ca(2+) 通道 Orai1 的结合，通过过表达 PIP5KIβ 而增强，并通过过表达 PIP5KIγ 而抑制。这些不同的 PIP5KI 同工型导致在去污剂抗性膜 (DRM) 部分中 PtdIns(4,5)P(2)的差异增强，该部分包含有序脂质区域和去污剂可溶膜 (DSM) 部分，包含无序脂质区域。这些结果一致表明，定位于有序脂质的肌醇 5-磷酸酶 L10-Inp54p 降低 DRM 部分的 PtdIns(4,5)P(2)并抑制 thapsigargin 刺激的 STIM1-Orai1 结合和储存操作的 Ca(2+)进入，而定位于无序脂质的肌醇 5-磷酸酶 S15-Inp54p 降低 DSM 部分的 PtdIns(4,5)P(2)并增强 STIM1-Orai1 结合。去除 STIM1 C 端多赖氨酸序列（氨基酸 677-685）或 Orai1 N 端多精氨酸序列（氨基酸 28-33）都消除了这种 STIM1-Orai1 结合对独特膜结构域中 PtdIns(4,5)P(2)的不同敏感性。我们的结果与 PtdIns(4,5)P(2) 平衡的模型一致，其中储存耗竭刺激的 STIM1-Orai1 结合受 PtdIns(4,5)P(2)的有序脂质池正向调节，并受无序脂质域中的 PtdIns(4,5)P(2)负向调节。
