CALCIUM CHANNELS IN NONEXCITABLE CELLS

在血管内皮细胞和各种非兴奋性细胞中， 类型，膜受体的刺激导致Ca2+从 内部存储和伴随的Ca2+从细胞外流入 空间虽然负责肌醇-1，4，5- 三磷酸盐（Ins（1，4，5）P3）诱导的Ca2+从内部储存释放是 与Ca2+内流相关的分子机制已经确立， 仍然未知。在许多细胞中，Ca2+的内流似乎是继发性的， 导致体内钙库的耗竭。 膜电流 已记录了由储存耗尽产生的Ca2+，并将其称为Ca2 + 释放激活电流或I-crac。 我们理解 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身份缺乏了解。 因此，本研究的长期目标是克隆和功能上 表达负责I-crac的蛋白质。一条线索来确认 途径，以及我们的克隆策略，来自对果蝇的研究， 光传导在一种叫做瞬时受体电位的突变果蝇中， trp是光传导级联中的缺陷，导致缩短的 强光刺激时的钙电流。trp编码的蛋白质， 和另一种与色氨酸同源的蛋白质，称为trpl，已经被提出， 是由Ins（1，4，5）P3依赖性的阳离子特异性通道激活的 Drosophila photoreceptor果蝇感光细胞生物化学相似性 果蝇的光转导和受体介导的Ca~（2+）信号转导 哺乳动物的非兴奋细胞表明，类似的蛋白质可能是 涉案我们的初步研究表明，trp和trpl是Ca2 + 可渗透阳离子通道，可被受体依赖性激活 机制等该项目的具体目标是： 机制，并确定trp和trpl的结构特征 与Ins（1，4，5）P3依赖性的通道活性调节相关 2）确定色氨酸孔内的氨基酸残基 和TRPL，它们是离子选择性的重要决定因素， 电导，和3）克隆和功能性表达的其他成员， 来自无脊椎动物和脊椎动物细胞的离子通道的Trp家族。到 为了实现这些目标，将 使用杆状病毒-Sf9细胞表达系统功能性表达。 离子选择性、电压敏感性和无机离子的阻断作用 将使用全细胞和单通道记录来确定阳离子。 色氨酸衍生的寡核苷酸探针将用于昆虫的筛选 和哺乳动物细胞cDNA文库。 全长同源克隆将是 表达和它们的功能特性相比，trp，trpl， 我...在内皮细胞中鉴定这种途径将是 特别重要的是，这些细胞在调节中起着重要作用， 血管张力和渗透性。

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.