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Ion Channel Regulation By Signal Transduction Pathways

通过信号转导途径调节离子通道

基本信息

批准号：
8336595
负责人：
David Armstrong
金额：
$ 111.34万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8336595
关键词：
Affinity Atosiban Biochemical Biological Assay Blood Vessels Brain Ca(2+)-Transporting ATPase Calcium Calcium Channel Calcium Signaling Cell Communication Cell Proliferation Cell physiology Cells Chemicals Chemosensitization Coupled Disease Endocrine Endocrine disruption Endoplasmic Reticulum Engineering Epithelial Flame Retardants Fluids and Secretions Fluorescence G-Protein-Coupled Receptors GTP-Binding Proteins Gene Expression Generations Genes Health Heart Rate Hippocampus (Brain)Homeostasis Hormone Receptor Hormones Human Human Cell Line Human body Hypothalamic structure Immune Ion Channel Ion Channel Protein Life Membrane Membrane Potentials Methods Molecular Mus Mutation Nature Neurons Neuropeptides Oxytocin Oxytocin Receptor Patch-Clamp Techniques Pathway interactions Pharmacologic Substance Phosphorylation Potassium Predisposition Reader Receptor Gene Recombinant Proteins Regulation Reporting Signal Pathway Signal Transduction Signal Transduction Pathway Slice Social Behavior Synapses System Thapsigargin Thyroid Hormones Time Toxic Environmental Substances Trust autism spectrum disorder base calcium indicator cell motility environmental chemical hippocampal pyramidal neuron human disease inorganic phosphate interest protein function receptor reproductive hormone toxicant voltage

项目摘要

To identify toxicants targeting oxytocin signaling, we engineered a human cell line (HEK293) to express human OXTR and a new generation, low affinity, genetically encoded calcium indicator, GCAMP3 (Tian, L. et al., 2009 Nature Methods 6:875-881). We screened the cells in a 96-well plate fluorescence reader. Calcium signaling was stimulated half maximally by 3 nM oxytocin and blocked half maximally by 60 nM atosiban, an established Oxtr antagonist. We identified two flame retardants, tris (2,3-dibromo-2-propyl) phosphate (TDBPP) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) that reduced the calcium signal when they were applied at concentrations between 20-100 uM. At the single cell level, however, atosiban and the flame retardants had very different effects on the calcium signals induced by oxytocin. The origin of the difference was revealed when we stimulated calcium release and entry independently of oxytocin by inhibiting the calcium pump in the endoplasmic reticulum (ER) with thapsigargin. As expected for an oxytocin receptor antagonist, atosiban had no effect on the calcium signals produced by thapsigargin. In contrast, both flame retardants selectively suppressed calcium entry after the ER calcium was depleted. The same concentrations of the flame retardants also inhibit the calcium-dependent synaptic potentiation induced by oxytocin on CA1 pyramidal neurons in hippocampal slices from PD 12-17 mouse brains. Thus, we conclude that synaptic potentiation by Gq-coupled hormone receptors requires calcium entry through store-operated calcium channels, such as ORAI2, which is highly expressed in hippocampal pyramidal neurons.

为了鉴定靶向催产素信号传导的毒物，我们工程化人细胞系（HEK 293）以表达人OXTR和新一代低亲和力遗传编码的钙指示剂GCAMP 3（Tian，L.例如，2009 Nature Methods 6：875-881）。我们在96孔板荧光读数器中筛选细胞。钙信号传导由3 nM催产素刺激一半最大值，并由60 nM阿托西班（一种已建立的Oxtr拮抗剂）阻断一半最大值。我们鉴定了两种阻燃剂，三（2，3-二溴-2-丙基）磷酸酯（TDBPP）和三（1，3-二氯-2-丙基）磷酸酯（TDCPP），当它们以20-100 μ M之间的浓度施用时，它们降低了钙信号。然而，在单细胞水平上，阿托西班和阻燃剂对催产素诱导的钙信号有非常不同的影响。当我们通过用毒胡萝卜素抑制内质网（ER）中的钙泵而独立于催产素刺激钙的释放和进入时，发现了这种差异的起源。正如催产素受体拮抗剂所预期的那样，阿托西班对毒胡萝卜素产生的钙信号没有影响。相反，这两种阻燃剂选择性地抑制钙进入ER钙耗尽后。相同浓度的阻燃剂还抑制由催产素对来自PD 12-17小鼠脑的海马切片中的CA 1锥体神经元诱导的钙依赖性突触增强。因此，我们得出结论，Gq偶联激素受体的突触增强需要钙进入通过存储操作的钙通道，如ORAI 2，这是高度表达在海马锥体神经元。