Characterization of a novel metal ion permeable channel

LTRPC7是一种新的广泛表达的二价离子通透通道，它在钙和镁的动态平衡和细胞活力中发挥着重要作用。它似乎也代表了锌和铁等必需痕量金属以及铝等有毒金属普遍存在的基本流入途径。鉴于必需金属在代谢过程中对生存至关重要的已知作用，以及越来越多的证据表明神经病理学中金属稳态的异常，建议对金属离子通过LTRPC7的渗透及其在正常和病理生理条件下的调节进行详细的研究。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。采用电生理方法和细胞内指示剂荧光成像相结合的方法，研究LTRPC7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原代培养中的功能及其调控。将通过指示剂染料的荧光成像和笼中猫的闪光光解以及神经细胞系NIE-115对M1毒扁豆碱激活的钙成像实验来研究细胞内钙对LTRPC7通道的调节。细胞内的镁和镁核苷酸对通道功能的调节将在潜在的镁2配位突变的表达的LTRPC7通道中被研究。将通过实验研究过氧化氢诱导的氧化应激对LTRPC7功能和金属离子渗透的影响，以评估该通道在病理生理条件下的行为。最后，为了确定神经退行性变的易感性是否由基因突变所致，将对LTRPC7基因的PCR扩增片段进行测序，并确定在关岛ALS和PD的脑组织中高发的多态现象。然后，这些突变将在表达系统中进行工程设计，以评估由此导致的通道功能变化。

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.