Molecular mechanisms of ER luminal [Ca2+] modulation of InsP3R channel activity

 描述（申请人提供）：普遍存在的内质网（ER）-定位的肌醇1，4，5-三磷酸（InsP 3）受体（InsP 3R）通道通过释放储存在ER腔中的Ca 2+以产生和调节控制许多生理过程的复杂Ca 2+信号来响应细胞外刺激而升高细胞质游离Ca 2+浓度（[Ca 2 +]i），从细胞凋亡和分泌到免疫反应和记忆。尽管胞质[InsP 3]和[Ca 2 +]i对InsP 3R通道活性的调节已被充分研究，但ER腔中游离[Ca 2 +]（[Ca 2 +]ER）的生理变化对InsP 3R通道活性的影响仍知之甚少且存在争议。更好地理解这些作用将有助于深入了解[Ca 2 +]ER稳态与InsP 3R调节的细胞内生理过程（包括细胞生物能量学和蛋白质生物合成的调节）的正常功能之间的联系;以及[Ca 2 +]ER失调与ER应激反应、糖尿病、心功能不全、细胞增殖缺陷等疾病的发病机制之间的联系 和细胞死亡。以往研究[Ca 2 +]ER对InsP 3R通道活性的影响主要采用45 Ca 2+通量测量和荧光Ca 2+成像，这两种方法都是从[Ca 2 +]i或[Ca 2 +]ER的变化来推断InsP 3R通道活性，因此不能同时严格控制[Ca 2 +]ER和[Ca 2 +]i。为了深入了解[Ca 2 +]ER对InsP 3R通道活性的调节，我们在初步研究中通过我们开创的核膜片钳方法在严格控制的离子条件下研究了单个InsP 3R通道的活性。单个InsP 3R通道的活动进行了连续监测，因为[Ca 2 +]ER通过快速灌注溶液开关或通过调节外核膜上的SERCA活性而改变。我们发现[Ca ~（2+）]ER通过两种不同的机制调节InsP_3R通道门控：（1）通过其胞浆内Ca ~（2+）敏感位点，驱动Ca ~（2+）通过开放通道，升高通道附近的局部[Ca ~（2+）]i，从而改变通道的活性;和（2）在许多细胞类型中，可能由定位于ER腔的外周辅助蛋白介导的显著的Ca 2+通量非依赖性效应。我们将应用单通道核电生理学和[Ca 2 +]i成像来系统地量化各种InsP 3R通道（重组和内源性，不同InsP 3R亚型的同源和异源四聚体）的各种[Ca 2 +]i，[InsP 3]和[Ca 2 +]ER中的每一种效应。此外，我们的初步研究表明，成员的膜联蛋白和copine家族的蛋白质存在于ER腔和相互作用，具体地说，在[Ca 2 +]ER依赖的方式，与ER腔片段保守的所有InsP 3R亚型。我们将验证，使用分子生物学和电生理学的方法，这些蛋白质的作用，在介导的Ca 2+流量独立的[Ca 2 +]ER对单个InsP 3R通道活动的影响，并在调节Ca 2+信号在细胞水平。这些研究将为InsP 3R通道活性的[Ca 2 +]ER调节提供新的见解，这似乎与更好地研究的[Ca 2 +]i通道调节一样深刻。

 DESCRIPTION (provided by applicant): The ubiquitous endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel raises cytoplasmic free Ca2+ concentration ([Ca2+]i) in response to extracellular stimuli by releasing Ca2+ stored in the ER lumen to generate and modulate complex Ca2+ signals that control numerous physiological processes, from apoptosis and secretion to immune responses and memory. Although regulation of InsP3R channel activity by cytoplasmic [InsP3] and [Ca2+]i has been well studied, the effects of physiological changes in free [Ca2+] in the ER lumen ([Ca2+]ER) on InsP3R channel activity remain poorly understood and controversial. Better understanding of such effects will provide insights into the connections between [Ca2+]ER homeostasis and proper functioning of InsP3R-modulated intracellular physiological processes including regulation of cell bioenergetics and protein biogenesis; and between [Ca2+]ER dysregulation and pathogenesis of conditions like ER stress responses, diabetes, cardiac dysfunction, defective cell proliferation and cell death. Previous studies of [Ca2+]ER effects on InsP3R channel activity used mostly 45Ca2+ flux measurement and fluorescence Ca2+ imaging, both of which infer InsP3R channel activity from changes in [Ca2+]i or [Ca2+]ER, and therefore cannot rigorously control both [Ca2+]ER and [Ca2+]i simultaneously. To provide insights into [Ca2+]ER modulation of InsP3R channel activity, we have investigated, in preliminary studies, single InsP3R channel activities under rigorously controlled ionic conditions by the nuclear patch clamp method that we pioneered. Activities of single InsP3R channels were continuously monitored as [Ca2+]ER was changed by rapid perfusion solution switches or by modulating activity of SERCA at the outer nuclear membrane. We have discovered that [Ca2+]ER modulates InsP3R channel gating by two distinct mechanisms: (1) driving Ca2+ flux through the open channel to raise local [Ca2+]i in the vicinity of the channel, thereby altering its activity through its cytoplasmic Ca2+-sensing sites; and (2) a profound Ca2+-flux-independent effect mediated likely by a peripheral accessory protein(s) localized to the ER lumen in many cell types. We will apply single-channel nuclear electrophysiology and [Ca2+]i imaging to systematically quantify each of these effects individually in various [Ca2+]i, [InsP3], and [Ca2+]ER for various kinds of InsP3R channels (recombinant and endogenous, homo- and hetero-tetrameric ones of different InsP3R isoforms). Furthermore, our preliminary studies demonstrated that members of the annexin and copine families of proteins are present in the ER lumen and interact specifically, in a [Ca2+]ER dependent manner, with a ER luminal fragment conserved in all InsP3R isoforms. We will verify, using molecular biological and electrophysiological methods the roles of these proteins in mediating the Ca2+-flux independent [Ca2+]ER effect on single InsP3R channel activities, and in regulating Ca2+ signaling at the cellular level. These studies will provide novel insights into [Ca2+]ER regulation of InsP3R channel activity, which appears to be as profound as the better studied [Ca2+]i regulation of the channel.