Molecular, genetic & physiological studies of calcium-activated chloride channels

分子、遗传

• 批准号: 9917848
• 负责人: LILY Y JAN
• 金额: $ 35.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9917848
• 关键词: Action Potentials; Address; Affect; Ambystoma; Anions; Automobile Driving; Binding; Binding Sites; Brain; Calcium; Calcium Channel; Calcium ion; Chemosensitization; Chloride Channels; Chloride Ion; Chlorides; Collaborations; Cryoelectron Microscopy; Cytosol; Dependence; Detergents; Development; Divalent Cations; Drosophila genus; Electrophysiology (science); Experimental Designs; Family; Feedback; Future; Hippocampus (Brain); Homologous Gene; Inferior; Integral Membrane Protein; Intervention; Iodides; Ions; Kinetics; Lead; Lipid Bilayers; Membrane; Membrane Potentials; Molecular; Molecular Conformation; Molecular Genetics; Mus; Mutagenesis; Mutate; N-Methyl-D-Aspartate Receptors; Names; Nervous system structure; Neurons; Oocytes; Pathway interactions; Physiological; Play; Property; Regulation; Reporting; Resolution; Role; Site-Directed Mutagenesis; Source; Structure; Synaptic Potentials; System; Therapeutic; Transcript; Transmembrane Domain; Work; Xenopus oocyte; base; biophysical analysis; biophysical techniques; constriction; experience; expression cloning; extracellular; fascinate; hippocampal pyramidal neuron; insight; member; motor learning; nanodisk; neurotransmission; novel; particle; prevent; reconstitution; voltage

Project Summary/Abstract To initiate molecular characterization of the calcium-activated chloride channels (CaCCs) that have been found in multiple neuronal types since 1980s, we first showed that CaCC is formed by TMEM16A or TMEM16B of the mammalian TMEM16 family of ten members in 2008. To ask how CaCC works, we investigated the calcium gating mechanism. First, we showed that a fruit fly homolog of the TMEM16 family, which we named Subdued, forms CaCC. Next, we mutated all 38 acidic residues that are evolutionarily conserved in fruit fly and mammalian CaCCs, to identify five acidic residues that strongly impact the calcium sensitivity of CaCC. After reporting our study, we are pleased to see that these five acidic residues correspond to the five acidic residues that bind two calcium ions in the recently reported structure of the fungal TMEM16 homolog, nhTMEM16. We will continue with our biophysical studies to elucidate how CaCC works by combining structural analyses of TMEM16A via single-particle electron cryo-microscopy (cryo-EM) with site- directed mutagenesis and electrophysiological studies. Having found CaCC involvement in the modulation of the action potential waveform and excitatory synaptic potentials in hippocampal neurons as well as action potential firing of inferior olivary neurons and cerebellar motor learning, we aim to conduct mechanistic studies to determine how CaCC works, in order to better understand CaCC modulation of neuronal signaling: Whereas it is well known that CaCC channel activity leads to membrane potential change, it is an intriguing open question as to how voltage across the membrane affects CaCC function. Whereas we know CaCC is activated by elevation of intracellular calcium that may result from calcium influx through calcium channels or NMDA receptors or calcium release from internal stores, it is unknown whether CaCC activation at low or high internal calcium concentration, which likely reflects different physiological contexts for CaCC modulation in neurons, might lead to the opening of different permeation pathways for chloride ions. Moreover, it is important to understand how chloride and other permeant ions such as iodide might exert feedback regulation of CaCC activity. Mechanistic understanding of CaCC function and modulation at the molecular level will not only provide insight as to how CaCC fulfills its physiological functions in the brain but also facilitate future development of CaCC modulators of potential therapeutic values.

项目总结/摘要 启动钙激活氯离子通道（CaCC）的分子表征， 自20世纪80年代以来，在多种神经元类型中发现，我们首次表明CaCC由TMEM 16 A或TMEM 16 B形成。 TMEM 16 B是哺乳动物TMEM 16家族的10个成员之一。为了了解CaCC如何工作，我们 研究了钙门控机制。首先，我们证明了果蝇TMEM 16家族的同源物， 我们称之为Subdued，形成CaCC。接下来，我们突变了所有38个酸性残基 在果蝇和哺乳动物CaCCs中保守，以确定五个强烈影响钙的酸性残基， CaCC的敏感性报告我们的研究后，我们高兴地看到，这五个酸性残基对应 在最近报道的真菌TMEM 16的结构中， 同源物，nhTMEM 16。我们将继续进行我们的生物物理研究，以阐明CaCC是如何工作的， 通过单粒子电子冷冻显微镜（cryo-EM）结合TMEM 16 A的结构分析， 定向诱变和电生理学研究。 发现CaCC参与了动作电位波形和兴奋性突触的调制， 海马神经元的动作电位以及下橄榄神经元和小脑的动作电位放电 运动学习，我们的目标是进行机制研究，以确定CaCC如何工作，以便更好地 了解神经元信号传导的CaCC调节：而众所周知，CaCC通道活性 导致膜电位变化，这是一个有趣的开放性问题， 影响CaCC功能。然而我们知道CaCC是通过细胞内钙离子的升高而激活的， 这是由于钙离子通过钙通道或NMDA受体内流或从内部释放钙引起的。 储存，目前还不清楚CaCC是否在低或高的内部钙浓度下活化，这可能 反映了神经元中CaCC调节的不同生理背景，可能导致不同的开放 氯离子的渗透途径。此外，重要的是要了解氯化物和其他 渗透性离子如碘离子可能对CaCC活性起反馈调节作用。机制理解 在分子水平上对CaCC功能和调节的研究不仅将提供关于CaCC如何实现 其在大脑中的生理功能也有利于未来开发CaCC调节剂的潜力 治疗价值。