CALCIUM CHANNELS IN NONEXCITABLE CELLS

非兴奋细胞中的钙通道

• 批准号: 2190867
• 负责人: WILLIAM P SCHILLING
• 金额: $ 20.2万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-11 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190867
• 关键词: Drosophilidae; G protein; Sf9 cell line; biological signal transduction; calcium channel; calcium flux; electrophysiology; inositol phosphates; membrane channels; molecular cloning; nucleic acid chemical synthesis; nucleic acid probes; oligonucleotides; photoactivation; polymerase chain reaction; protein sequence; protein structure function; receptor binding; single cell analysis; tissue /cell culture; vascular endothelium; voltage /patch clamp

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.

在血管内皮细胞中，在各种非驱散细胞中 类型，刺激膜受体会导致Ca2+从 内部存储和伴随细胞外Ca2+的涌入 空间。尽管负责肌醇L的机制，但4,5-- trisphophate（INS（L，4,5）P3）诱导的Ca2+从内部商店释放为 良好的，与Ca2+流入相关的分子机制 仍然未知。在许多细胞中，Ca2+的流入似乎是次要的 内部Ca2+存储的耗竭。 膜电流 已记录并将其称为CA2+产生的。 释放激活的电流或I-Crac。 我们了解 与I-CRAC激活相关的生化机制受到阻碍 我们缺乏有关该途径分子身份的知识。 因此，这项研究的远距离目标是克隆并在功能上 表达负责I-Crac的蛋白质。对此身份的线索 途径和我们的克隆策略源自果蝇的研究 光转导。在称为瞬态受体电位的突变蝇中，或 TRP，光转移级联的缺陷导致缩写 在强烈的光刺激中，Ca2+电流。由TRP编码的蛋白质， 并且已经提出了另一种称为TRPL的蛋白质的蛋白质 由INS（L，4,5）P3依赖性激活的特定阳离子通道 果蝇光感受器的机理。之间的生化相似性 果蝇和受体介导的Ca2+信号传导的光转传 哺乳动物的非脱离细胞表明类似蛋白可能是 涉及。我们的初步研究表明，TRP和TRPL为Ca2+ 可渗透的阳离子通道可以被受体依赖激活 机制。该项目的具体目的是确定 机理并确定TRP和TRPL的结构特征 与INS（L，4,5）P3依赖性调节通道活性有关 机制，2）鉴定TRP孔内的氨基酸残基 和trpl是离子选择性的重要决定因素和 电导和3）克隆并在功能上表达其他成员 来自无脊椎动物和脊椎动物细胞的离子通道的TRP家族。到 完成这些目标，TRP，TRPL和嵌合通道结构将是 使用杆状病毒-SF9细胞表达系统功能表达。 离子选择性，电压敏感性和无机封锁 阳离子将使用全电池和单个通道记录确定。 从TRP得出的寡核苷酸探针将用于筛查昆虫 和哺乳动物细胞cDNA库。 全长同源克隆将是 与TRP，TRPL和 i-crac。在内皮细胞中识别该途径将是 特别重要，因为这些细胞在调节中起着重要的作用 血管张力和渗透性。