Characterization of a novel metal ion permeable channel

LTRPC7是一种新型的广泛表达的二价离子渗透通道，在Ca2+和Mg2+稳态和细胞活力中发挥重要作用。它似乎也代表了基本微量金属（如Zn2+和Fe2+）以及有毒金属（如Al3）普遍存在的基础流入途径。鉴于已知必需金属在对生存至关重要的代谢过程中的作用，以及越来越多的证据表明金属稳态异常在神经病理学中存在，我们提出了一项通过LTRPC7的金属离子渗透及其在正常和病理生理条件下的调节的详细研究。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。我们将采用电生理方法和细胞内指示染料的荧光成像相结合的方法，研究LTRPC7在HEK-293表达系统、神经元和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。我们将利用指示染料的荧光成像和笼型Cat+的闪光光解，以及M1毒菌碱激活后神经元细胞系NIE-115的钙成像实验，研究细胞内Ca2+对LTRPC7通道的调控。在潜在的Mg2+配位位点发生突变的表达LTRPC7通道中，将研究细胞内Mg +和Mg-核苷酸对通道功能的调节。通过实验研究h2o2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，为了确定神经变性的易感性是否与基因突变有关，将对pcr扩增的LTRPC7基因片段进行测序，并鉴定出在关岛ALS和PD脑组织中高发的多态性。然后将这些突变在表达系统中进行工程化，以评估由此产生的通道功能改变。

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.