Characterization of a novel metal ion permeable channel

新型金属离子渗透通道的表征

• 批准号: 6726086
• 负责人: MEREDITH C HERMOSURA
• 金额: $ 31.98万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726086
• 关键词: astrocytes; biological signal transduction; calcium ion; cell line; gene mutation; genetic polymorphism; glia; human tissue; imaging /visualization /scanning; ion transport; iron; magnesium ion; membrane channels; minority institution research support; neural degeneration; neurons; neurophysiology; northern blottings; oxidative stress; polymerase chain reaction; postmortem; protein structure function; voltage /patch clamp; zebrafish; zinc

LTRPC7 is a novel widely expressed divalent ion-permeable channel that appears to play an important role in Ca2+ and Mg2+ homeostasis and in cell viability. It also appears to represent a ubiquitous basal influx pathway for essential trace metals like Zn2+ and Fe2+ as well as toxic metals such as Al3. Given the known role of essential metals in metabolic processes vital for survival and mounting evidence implicating aberrations of metal homeostasis in neuropathology, a detailed study of metal ion permeation through LTRPC7 and its regulation in normal and pathophysiological conditions, is proposed. The study will be conducted as a collaboration between investigators at the University of Hawaii and Stanford University. An integrated approach using electrophysiological methods and fluorescence imaging of intracellular indicator dyes will be used to study LTRPC7 function and its regulation in the HEK-293 expression system, neuronal and glial cell lines, and primary cultures of zebrafish cerebellar neurons. Regulation of LTRPC7 channels by intracellular Ca2+ will be studied using fluorescence imaging of indicator dyes and flash photolysis of caged Cat+, as well as calcium imaging experiments in the neuronal cell line NIE-115 upon M1 muscarinic activation. The regulation of channel function by intracellular Mg 2+, and Mg-- nucleotides will be investigated in expressed LTRPC7 channels where potential Mg2+ coordination sites have been mutated. Experiments to study the effects of H2O2-induced oxidative stress on LTRPC7 function and metal ion permeation will be conducted to assess how the channel behaves during pathophysiological conditions. Finally, to determine whether susceptibility to neurodegeneration is conferred by genetic mutations, PCR-amplified fragments of the LTRPC7 gene will be sequenced and polymorphisms occurring with high incidence in brain tissue of Guamanian ALS and PD identified. These mutations will be then engineered in the expression system to assess resulting alterations in channel function.

LTRPC 7是一种新的广泛表达的二价离子渗透通道，似乎在Ca 2+和Mg 2+稳态和细胞活力中发挥重要作用。它似乎也代表了一个普遍存在的基础流入途径的必需微量金属，如锌2+和铁2+以及有毒金属，如铝。鉴于已知的作用，必需金属在代谢过程中至关重要的生存和越来越多的证据暗示畸变的金属稳态神经病理学，金属离子渗透通过LTRPC 7和它的调节在正常和病理生理条件下，提出了一个详细的研究。这项研究将由夏威夷大学和斯坦福大学的研究人员合作进行。使用电生理学方法和细胞内指示染料的荧光成像的综合方法将用于研究LTRPC 7功能及其在HEK-293表达系统、神经元和神经胶质细胞系以及斑马鱼小脑神经元的原代培养物中的调节。将使用指示剂染料的荧光成像和笼状Cat+的闪光光解，以及M1毒蕈碱激活后神经元细胞系NIE-115中的钙成像实验，研究细胞内Ca 2+对LTRPC 7通道的调节。在表达的LTRPC 7通道中，潜在的Mg 2+配位位点已经突变，将研究细胞内Mg 2+和Mg-核苷酸对通道功能的调节。将进行研究H2 O2诱导的氧化应激对LTRPC 7功能和金属离子渗透的影响的实验，以评估通道在病理生理条件下的行为。最后，为了确定神经变性的易感性是否由基因突变赋予，将对LTRPC 7基因的PCR扩增片段进行测序，并鉴定在关岛ALS和PD脑组织中发生率高的多态性。然后将这些突变在表达系统中进行工程改造，以评估通道功能的改变。