Mapping neuronal chloride microdomains

绘制神经元氯微区

• 批准号: 8822651
• 负责人: Kevin J. Staley
• 金额: $ 91.33万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-26 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822651
• 关键词: Anions; Area; Biological Neural Networks; Biology; Biopolymers; Brain; Calcium; Chloride Ion; Chlorides; DNA; Dependence; Engineering; Extracellular Matrix; Face; Family; Funding; Funding Mechanisms; Genetic; Glutamates; In Situ; In Vitro; Inorganic Sulfates; Interneurons; Laboratories; Ligands; Link; Location; Maps; Measures; Mediating; Membrane; Membrane Protein Traffic; Memory; Methods; Microtubules; Mus; Neurons; Neurophysiology - biologic function; Noise; Output; Process; Proteins; Qualifying; RNA; Reading; Reporter; Reporting; Resolution; Series; Side; Signal Transduction; Structure; Synapses; System; Techniques; Testing; Time; Transgenic Mice; Tubulin; Unspecified or Sulfate Ion Sulfates; analog; base; brain volume; clomeleon; extracellular; fluorophore; gamma-Aminobutyric Acid; in vivo; insight; novel strategies; operation; parallel processing; polyanion; polysulfated glycosaminoglycan; public health relevance; ratiometric; receptor; research study; sensor; transgene expression

 DESCRIPTION (provided by applicant): Dramatic new insights into the functioning of neural networks have been made possible by our ability to visualize neural function with calcium-sensitive fluorophores, and biology has been revolutionized by the ability to sequence and manipulate DNA, RNA, and proteins. Both of these tremendous advances have unexplored "flip sides". Our understanding of neural network function remains limited by our inability see GABA-mediated synaptic activity: we can't measure the output of the remarkable diversity of interneuron structure and function. Similarly, the methods for studying templated biopolymers such as DNA, RNA, and protein are not applicable to untemplated biopolymers such as polyglutamylated intracellular tubulin, and the variably sulfated glycosaminoglycans (GAGs) that comprise the extracellular matrix. The glutamate and sulfate moieties displace chloride, thereby defining chloride microdomains. These microdomains thus provide a "photographic negative" of the information that may be stored in local untemplated biopolymers. Several compelling lines of evidence suggest that chloride microdomains alter the direction and magnitude of currents flowing through open GABAA receptors, providing a mechanism to read out information stored in the anion distribution of the biopolymers. This implies a unique direction and size of chloride current at each GABAA receptor, so that these critical determinants must be measured to understand the contribution of interneurons to network operation. To visualize chloride microdomains, we have assembled a uniquely qualified team to create and fully characterize a series of reporters of chloride concentration placed at the most critical location: the intra and extracellular faces of GABAA receptor subunits. Super Clomeleon, a new ratiometric chloride- sensitive fluorophore, will be fused to g2 and d subunits of the GABAA receptor. g2-linked Super Clomeleon will report synaptic chloride microdomains that drive synaptic GABA signaling, while d-linked Super Clomeleon will report chloride domains subserving extrasynaptic GABA signaling. Both intra and extracellular chloride microdomains will be analyzed by the Super Clomeleon - GABAA subunit fusion constructs, so that the direction and amplitude of chloride currents can be precisely mapped. The GABAA subunit-fluorophore fusion products will be parallel-processed in four labs so that in the three- year time window of this RFA we can assess receptor assembly, membrane trafficking, receptor conductance, chloride sensitivities, gains, pH dependence, and stability in vitro and in vivo. We can then make timely go / no go decisions regarding the utility of going forward to create transgenic mice.

 描述（由申请人提供）：通过我们使用钙敏感荧光团可视化神经功能的能力，对神经网络功能的戏剧性新见解已经成为可能，并且生物学已经通过测序和操纵DNA，RNA和蛋白质的能力而发生了革命性变化。这两个巨大的进步都有未被探索的“反面”。我们对神经网络功能的理解仍然受到我们无法看到GABA介导的突触活动的限制：我们无法测量中间神经元结构和功能的显着多样性的输出。类似地，用于研究模板化的生物聚合物如DNA、RNA和蛋白质的方法不适用于非模板化的生物聚合物如聚谷氨酰化的细胞内微管蛋白和包含细胞外基质的硫酸化糖胺聚糖（GAG）。谷氨酸盐和硫酸盐部分取代氯，从而定义氯微域。因此，这些微区提供了可以存储在局部非模板化生物聚合物中的信息的“照相底片”。一些令人信服的证据表明，氯微区改变了流经开放GABAA受体的电流的方向和幅度，提供了一种读出存储在生物聚合物阴离子分布中的信息的机制。这意味着每个GABAA受体的氯电流的方向和大小都是独一无二的，因此必须测量这些关键决定因素，以了解中间神经元对网络运行的贡献。为了可视化氯离子微区，我们组建了一个独特的合格团队，创建并充分表征了一系列位于最关键位置的氯离子浓度报告分子：GABAA受体亚基的细胞内和细胞外表面。超级克隆龙，一种新的比率氯敏感荧光团，将融合到GABAA受体的g2和d亚基。g2连接的超级克隆龙将报告驱动突触GABA信号传导的突触氯微结构域，而d连接的超级克隆龙将报告辅助突触外GABA信号传导的氯结构域。通过Super Clomeleon - GABAA亚基融合构建体分析细胞内和细胞外氯离子微结构域，从而可以精确绘制氯离子电流的方向和幅度。GABAA亚基-荧光团融合产物将在四个实验室中平行处理，以便在该RFA的三年时间窗内，我们可以评估受体组装、膜运输、受体电导、氯离子敏感性、增益、pH依赖性和体外和体内稳定性。然后，我们可以及时做出关于继续创造转基因小鼠的实用性的决定。