CALCIUM CHANNELS IN NONEXCITABLE CELLS

在血管内皮细胞中，以及在各种非兴奋细胞中 膜受体的类型，刺激引起钙离子的释放 细胞内储存和伴随的细胞外钙离子内流 太空。尽管肌醇的作用机制-L，4，5- 三磷酸(INS(L，4，5)P3)诱导的细胞内钙释放 众所周知，与钙离子内流相关的分子机制 仍然不为人所知。在许多细胞中，钙离子的内流似乎是次要的 与体内钙库的耗尽有关。膜电流 由于储存耗尽而产生的钙离子已经被记录下来，并被称为钙离子 释放激活电流，或I-Crac。我们理解世界的能力 与I-Crac激活相关的生化机制受到以下因素的阻碍 我们对这一途径的分子特性缺乏了解。 因此，这项研究的长期目标是克隆和功能 表达与i-Crac有关的蛋白。这是关于这一事件的身份的线索 途径，以及我们的克隆策略，源于对果蝇的研究 光转导。在一种名为瞬时受体潜力的突变果蝇中，或 Trp，光传导级联中的缺陷导致缩写 强光刺激时的钙电流。色氨酸编码的蛋白质， 而另一种与色氨酸同源的蛋白质，称为TRPL，已被提出用于 依赖INS(L，4，5)P3激活的Be阳离子特异性通道 果蝇光感受器的机制。它们在生物化学上的相似性 果蝇的光信号转导和受体介导的钙信号转导 哺乳动物的不可兴奋细胞表明，类似的蛋白质可能是 牵涉其中。我们的初步研究表明，色氨酸和色氨酸是钙离子 可被受体依赖性激活的通透性阳离子通道 机械装置。这个项目的具体目标是L)来确定 机制(S)，并确定Trp和Trpl的结构特征 INS(L，4，5)P3依赖对通道活动的调节 机理，2)识别Trp孔内的氨基酸残基 和TRPL是离子选择性的重要决定因素 传导性，以及3)克隆和功能表达 无脊椎动物和脊椎动物细胞中的Trp离子通道家族。至 为了实现这些目标，TRP、TRPL和嵌合通道结构将 利用杆状病毒-Sf9细胞表达系统进行功能表达。 无机盐对离子的选择性、电压敏感性和阻隔 阳离子将使用全细胞和单通道记录来确定。 从色氨酸衍生的寡核苷酸探针将用于昆虫筛选 和哺乳动物细胞cDNA文库。全长同源克隆将是 及其与Trp、Trpl和Trp的功能特性比较 我-克雷克。在内皮细胞中鉴定这一途径将是 尤其重要的是这些细胞在调节中起着突出的作用 血管紧张度和通透性。

In vascular endothelial cells, and in a variety of non-excitable cell types, stimulation of membrane receptors causes the release of Ca2+ from internal stores and the concomitant influx of Ca2+ from the extracellular space. Although the mechanisms responsible for inositol- l,4,5- trisphosphate(Ins(l,4,5)P3)-induced Ca2+ release from internal stores are well established, the molecular mechanisms associated with Ca2+ influx remain unknown. In many cells, the influx of Ca2+ appears to be secondary to the depletion of the internal Ca2+ store. The membrane current generated by depletion of the store has been recorded and termed the Ca2+ release-activated current, or I-crac. Our ability to understand the biochemical mechanisms associated with activation of I-crac is hampered by our lack of knowledge concerning the molecular identity of this pathway. Thus, the long-range goal of this study is to clone and functionally express the protein responsible for I-crac. A clue to the identity of this pathway, and our cloning strategy, derives from studies of Drosophila phototransduction. In a mutant fly called transient receptor potential, or trp, a defect in the phototransduction cascade results in an abbreviated Ca2+ current during intense light stimulation. The protein encoded by trp, and another protein homologous to trp, called trpl, have been proposed to be cation specific channels activated by an Ins(l,4,5)P3-dependent mechanism in Drosophila photoreceptor. The biochemical similarity between phototransduction in Drosophila and receptor-mediated Ca2+ signaling in mammalian non-excitable cells suggests that similar proteins may be involved. Our preliminary studies have shown that trp and trpl are Ca2+ permeable cation channels that can be activated by receptor-dependent mechanisms. The specific aims of this project are l) to determine the mechanism(s) and identify the structural features of trp and trpl associated with regulation of channel activity by Ins(l,4,5)P3-dependent mechanisms, 2) to identify the amino acid residues within the pore of trp and trpl that are important determinants of ionic selectivity and conductance, and 3) to clone and functionally express other members of the trp family of ion channels from both invertebrate and vertebrate cells. To accomplish these aims, trp, trpl, and chimeric channel constructs will be functionally expressed using the baculovirus-Sf9 cell expression system. The ion selectivity, voltage-sensitivity, and blockade by inorganic cations will be determined using whole cell and single channel recordings. Oligonucleotide probes derived from trp will be used for screening insect and mammalian cell cDNA libraries. Full length homologous clones will be expressed and their functional characteristics compared to trp, trpl, and I-crac. Identification of this pathway in endothelial cells will be particularly important as these cells play a prominent role in regulation of vascular tone and permeability.