Department of Biophysics, Saarland University, D-66421 Homburg, Germany.
J Biol Chem. 2013 Jan 18;288(3):1653-64. doi: 10.1074/jbc.M112.417246. Epub 2012 Dec 4.
A drop of endoplasmic reticulum Ca(2+) concentration triggers its Ca(2+) ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca(2+) entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca(2+) homeostasis.
内质网 Ca(2+) 浓度的下降会触发其 Ca(2+) 感应器蛋白基质相互作用分子 1（STIM1）寡聚化并在内质网-质膜连接处积聚，在该处它激活 Orai1 通道，提供储存操纵的 Ca(2+) 内流。为了阐明 STIM1 的 N-糖基化位点的功能意义，我们创建了不同的天冬酰胺-131 和天冬酰胺-171 的突变。STIM1 NN/DQ 导致功能获得性增强。膜片钳、全内反射荧光（TIRF）显微镜和荧光恢复后漂白（FRAP）分析表明，STIM1 DQ 突变体的表达增加了活性 Orai1 通道的数量和 STIM1 向内质网-质膜连接处易位的速度，而电流潜伏期缩短。令人惊讶的是，STIM1 DQ 的共表达降低了 Orai1 蛋白，改变了 STIM1:Orai1 比例。我们描述了一种新的数学工具，用于从比例变化中区分改变的 STIM1 或 Orai1 扩散参数的影响。该突变揭示了一种新的机制，即“超活性”STIM1 DQ 导致寡聚化速率常数改变，并导致 Orai1 降解，同时改变激活剂（STIM1）与效应物（Orai1）的比例，从而导致 Ca(2+) 稳态改变。
