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：
8929757
负责人：
David Armstrong
金额：
$ 86.67万
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/8929757
关键词：
Acute Affinity Appearance Atosiban Biochemical Biological Assay Biological Models Blood Vessels Brain Ca(2+)-Transporting ATPase Calcium Calcium Signaling Cell Communication Cell Proliferation Cell physiology Cells Chemicals Chemosensitization Cyclic AMP Disease Endocrine Endocrine disruption Endoplasmic Reticulum Engineering Epithelial Flame Retardants Fluids and Secretions Fluorescence G Protein-Coupled Receptor Genes G-Protein-Coupled Receptors GTP-Binding Proteins Gene Expression Generations Genes Health Heart Rate Heterotrimeric GTP-Binding Proteins Hippocampus (Brain)Homeostasis Hormones Human Human Cell Line Human body Hypothalamic structure Immune Ion Channel Ion Channel Protein Knockout Mice Life Membrane Membrane Potentials Methods Molecular Mouse Strains Mus Mutation Nature Neurons Neuropeptides Oxytocin Oxytocin Receptor Patch-Clamp Techniques Pathway interactions Pharmacologic Substance Phosphatidylinositol 4,5-Diphosphate Phosphorylation Potassium Potassium Channel Predisposition Protein phosphatase Reader Receptor Gene Recombinant Proteins Recombinants Regulation Reporting Rodent Signal Pathway Signal Transduction Signal Transduction Pathway Slice Social Behavior Somatostatin Somatostatin Receptor 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 reconstitution 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. We have subsequently shown that the flame retardants block TRPC5 channels, which were thought to be regulated by Gq signaling through calcium and PIP2 and/or its metabolites. However, the flame retardants do not interfere with Gq signaling or calcium release. We discovered that they block TRPC5 channels completely, and not by blocking the pore directly. We also showed that TRPC5 is required for oxytocin-dependent potentiation because it is missing in a TRPC5 knockout mouse strain. We are now examining the mechanism of TRPC5 stimulation by Gq signaling with recombinant channels in a heterologous system mammalian system. We have also established that somatostatin stops spiking through a diffusible, cAMP-dependent mechanism that targets two pore potassium channels involving protein phosphatases and we have reconstituted this signaling in a heterologous system. We have confirmed this mechanism in rodent hippocampal neurons, both in acute slices and in dissociated cultures. In fact there are two mechanisms in the neurons, the appearance of one of which is also regulated by protein phosphatases.

为了鉴定靶向催产素信号传导的毒物，我们设计了人类细胞系（HEK293）来表达人类OXTR和新一代，低亲和力，遗传编码的钙指标，GCAMP3（Tian，L。等，2009自然方法6：875-881）。我们在96孔板荧光读取器中筛选了细胞。钙信号传导被3 nm的催产素最大程度地刺激了一半，并被已建立的Oxtr拮抗剂Atosiban 60 nm Atosiban最大化。我们确定了两种阻燃剂，即Tris（2,3-二溴丙基）磷酸盐（TDBPP）和Tris（1,3-二氯2-丙酰基）磷酸盐（TDCPP），当钙信号降低时，它们在浓度之间以20-100 um的浓度施加时降低了钙信号。然而，在单细胞水平上，阿托西子和火焰阻燃剂对催产素诱导的钙信号的影响非常不同。当我们通过用thapsigargin抑制内质网（ER）中的钙泵来独立于催产素刺激钙释放和进入时，差异的起源被揭示了。正如催产素受体拮抗剂所预期的那样，Atosiban对Thapsigargin产生的钙信号没有影响。相比之下，两种阻燃剂在耗尽ER钙后有选择地抑制钙进入。相同浓度的阻燃剂还抑制了催产素在PD 12-17小鼠大脑的海马切片中催产素诱导的钙依赖性突触增强。随后，我们表明，火焰阻燃剂阻断了TRPC5通道，这些通道被认为是通过钙和PIP2和/或其代谢产物来调节的。但是，阻燃剂不会干扰GQ信号传导或钙释放。我们发现它们完全阻止了TRPC5通道，而不是直接阻止孔。我们还表明，TRPC5是催产素依赖性增强所必需的，因为它在TRPC5敲除小鼠应变中缺少。现在，我们正在研究异源哺乳动物系统中的重组通道通过GQ信号传导刺激TRPC5刺激的机制。我们还确定，生长抑素通过一种可扩散的，依赖的cAMP依赖性机制停止尖峰，该机制靶向涉及蛋白质磷酸酶的两个孔钾通道，并且我们在异源系统中重新安置了该信号。我们已经在急性切片和解离培养物中证实了啮齿动物海马神经元中的这种机制。实际上，神经元中有两种机制，其中一种机制也受蛋白质磷酸酶调节。