Bhardwaj Rajesh, Augustynek Bartłomiej S, Ercan-Herbst Ebru, Kandasamy Palanivel, Seedorf Matthias, Peinelt Christine, Hediger Matthias A
Membrane Transport Discovery Lab, Department of Nephrology and Hypertension, Inselspital Bern, Bern, Switzerland,
Department of Biomedical Research, University of Bern, Bern, Switzerland.
Cell Physiol Biochem. 2020 Mar 17;54(2):252-270. doi: 10.33594/000000218.
BACKGROUND/AIMS: Store-operated Ca entry (SOCE) through plasma membrane Ca channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca release-activated Ca current (I). A major regulatory mechanism that prevents deleterious Ca overload is the slow Ca-dependent inactivation (SCDI) of I. Several studies have suggested a role of Ca/calmodulin (Ca/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca/CaM-mediated SCDI of I is as yet unclear.
The Ca/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology.
Here we identified residues Leu/Val (H1) and Leu/Phe (H2) within SOAR that serve as hydrophobic anchor sites for Ca/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca/CaM binding. Single residue mutations of Phe to less hydrophobic residues significantly diminished SOCE and I, independent of Ca/CaM. Hence, the role of H2 residues in Ca/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of I caused by Ca/CaM, proving a direct role of STIM1 in coordinating SCDI.
Taken together, we propose a mechanistic model, wherein binding of Ca/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca/CaM are functionally coordinated to control I.
背景/目的：通过质膜钙通道Orai1的储存式钙内流（SOCE）对许多细胞过程至关重要。SOCE由内质网（ER）钙储存耗竭激活，依赖于ER锚定的钙传感蛋白STIM1的Orai1激活区域SOAR的门控功能。在电生理学上，SOCE的特征为钙释放激活钙电流（I）。防止有害钙超载的一种主要调节机制是I的缓慢钙依赖性失活（SCDI）。多项研究表明钙/钙调蛋白（Ca/CaM）在触发SCDI中起作用。然而，STIM1在调节Ca/CaM介导的I的SCDI中的直接作用尚不清楚。
通过在钙调蛋白琼脂糖珠上拉下重组绿色荧光蛋白（GFP）标记的人STIM1 C末端片段来测试Ca/CaM与STIM1的结合。在稳定过表达人Orai1的HEK293细胞中，利用CRISPR/Cas9技术敲除STIM1。使用荧光成像板读数器和全细胞膜片钳测量转染了STIM1钙调蛋白结合突变体的细胞中的储存式钙内流。在电生理学实验中，通过在膜片电极中加入重组人钙调蛋白来研究Ca/CaM在SCDI中的作用。
在此我们确定了SOAR内的亮氨酸/缬氨酸（H1）和亮氨酸/苯丙氨酸（H2）残基作为Ca/CaM结合的疏水锚定位点。双功能的H2位点对Orai1激活和Ca/CaM结合均至关重要。苯丙氨酸向疏水性较低的残基的单残基突变显著减少了SOCE和I，与Ca/CaM无关。因此，无法精确评估H2残基在Ca/CaM介导的SCDI中的作用。相比之下，H1位点仅控制Ca/CaM结合以及随后的SCDI，但不控制Orai1激活。V375A而非V375W替代消除了由Ca/CaM引起的I的SCDI，证明了STIM1在协调SCDI中的直接作用。
综上所述，我们提出了一个机制模型，其中Ca/CaM与STIM1疏水锚定残基H1和H2的结合通过破坏STIM1与Orai1之间的功能相互作用来触发SCDI。我们的研究结果揭示了STIM1、Orai1和Ca/CaM如何在功能上协同控制I。
