Structural and functional studies of CALHM channels

CALHM通道的结构和功能研究

• 批准号: 10350691
• 负责人: Wei Lu
• 金额: $ 47.33万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10350691
• 关键词: Adenosine Triphosphate; Alzheimer' s Disease; Anions; Architecture; Behavior; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Brain; Brain Injuries; Brain Ischemia; Calcium; Cells; Chemosensitization; Complex; Connexins; Consensus; Cryoelectron Microscopy; Data; Dependence; Detergents; Development; Dyes; Electrophysiology (science); Environment; Exocytosis; Family; Family member; Foundations; Gap Junctions; Hearing; Homeostasis; Human; In Vitro; Inflammation; Ion Channel; Ions; Ischemic Brain Injury; Knowledge; Ligand Binding; Link; Lipids; Mediating; Membrane; Mental Depression; Methods; Mission; Molecular; Molecular Conformation; Molecular Structure; Mutagenesis; Mutate; Mutation; N-terminal; Nervous System Physiology; Nervous system structure; Neurodegenerative Disorders; Neurons; Nociception; Pathologic; Permeability; Pharmacology; Physiology; Play; Polymers; Process; Property; Public Health; Publishing; Reporting; Research; Role; Ruthenium Red; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Solid; Structure; Synapses; Synaptic Cleft; Synaptic Transmission; Taste Buds; Taste Perception; Therapeutic Agents; United States National Institutes of Health; Work; antagonist; density; experimental study; extracellular; family structure; in vivo; insight; member; mutant; nervous system disorder; neuronal excitability; neuroprotection; novel therapeutics; particle; patch clamp; preservation; receptor; sensor; small molecule; stoichiometry; therapeutic development; therapeutic target; transmission process; voltage

PROJECT SUMMARY Purinergic signaling plays fundamental roles in activities of the nervous system as diverse as neuroprotection, synaptic transmission, nociception, inflammation, hearing, and taste. This process is initiated by releasing adenosine triphosphate (ATP) across the membrane through either classic exocytosis or ATP-permeable channels into the synaptic cleft, where the ATP binds downstream receptors on an adjacent cell. There are five families of ATP release channels: connexins, pannexins, volume-regulated anion channels, maxi channels, and calcium homeostasis modulators (CALHMs). Highly expressed in the brain and taste buds, CALHM channels play essential roles in taste and neuron transmission, and their dysregulation has been associated with various neurological disorders including Alzheimer disease, ischemic brain damage, and depression, making CALHM channels important pharmacological targets. The CALHM family consists of three members, CALHM1, 2, and 3. They are voltage-dependent, extracellular, calcium-concentration-regulated, nonselective ion channels that are permeable to the signaling molecules ATP and calcium. They are predicted to share membrane topology with connexins, pennexins, innexins and VRACs. Functional studies provide a consensus view that CALHM1 forms a hexameric channel and that it forms only hemichannels, but not gap junctions. CALHMs activity is modulated by a wide range of factors including ruthenium red, Gd3+, and 2-APB. Although CALHMs are central to human physiology and are potential therapeutic targets, there are no structures of this family. We do not understand, in molecular detail, how the channel is activated or inhibited, or how it is modulated by small molecules binding at specific sites. We have published strong evidence that CALHM2 is undecameric and exists as both hemichannels and gap junctions in vitro. We have determined cryo-EM structures of human CALHM2 in the Ca2+-free open state, and ruthenium red–bound inhibited state. These preliminary results provide not only the first atomic structures of a CALHM family member, but also the first bona fide structure in an inhibited state, which has never been reported for channels with similar topology including connexins, pannexins, innexins and VRACs. We observed a binding site of ruthenium red that was completely unknown before. Building on this preliminary data, we propose to continue the structural studies of CALHM2 and the other two family members, CALHM1 and CALHM3, combined with complementary electrophysiology experiments and other functional approaches, to define the molecular basis for a comprehensive gating mechanism and the molecular determinants and function of gap junction formation, as well as their pharmacology. These advances will provide a solid foundation for developing new drugs against neurodegenerative diseases and for a deeper understanding of the function of the ATP release channel family and the gap junction family.

项目摘要 嘌呤能信号传导在神经系统活动中起着重要作用， 突触传递、伤害感受、炎症、听觉和味觉。这一过程是通过释放 三磷酸腺苷（ATP）通过经典的胞吐作用或ATP渗透性 通道进入突触间隙，在那里ATP结合相邻细胞上的下游受体。有五 ATP释放通道家族：连接蛋白、泛连接蛋白、体积调节阴离子通道、maxi通道和 钙稳态调节剂（calcium homeostasis modulators，CALHMs）。在大脑和味蕾中高度表达，CALHM通道 在味觉和神经元传递中起重要作用，它们的失调与各种 神经系统疾病，包括阿尔茨海默病，缺血性脑损伤和抑郁症，使CALHM 通道重要的药理学靶点。 CALHM家族由三个成员组成，CALHM 1、2和3。它们是电压依赖性的，细胞外的， 钙浓度调节的非选择性离子通道，可渗透信号分子ATP 和钙。预测它们与连接蛋白、pennexin、连接蛋白和VRAC共享膜拓扑结构。 功能研究提供了一个共识，即CALHM 1形成六聚体通道，并且它只形成 半通道，而不是间隙连接。CALHMs的活性受多种因素的调节，包括 钌红、Gd 3+和2-APB。虽然CALHM是人类生理学的核心， 治疗靶点，没有这个家族的结构。我们不知道，在分子的细节， 通道被激活或抑制，或者它是如何通过在特定位点结合的小分子来调节的。 我们已经发表了强有力的证据，CALHM 2是十一聚体，并存在于半通道和间隙 体外连接。我们已经确定了人CALHM 2在无钙开放状态下的冷冻电镜结构， 钌红结合抑制状态。这些初步结果不仅提供了第一个原子结构的一个 CALHM家族成员，也是第一个真正处于抑制状态的结构，这从未有过报道 适用于具有相似拓扑结构的通道，包括连接蛋白、泛连接蛋白、连接蛋白和VRAC。我们观察到 钌红的位置，这是完全未知的。根据这些初步数据，我们建议 继续研究CALHM 2和其他两个家族成员CALHM 1和CALHM 3的结构， 与补充电生理学实验和其他功能的方法，以确定分子 全面门控机制的基础和间隙连接的分子决定因素和功能 形成，以及它们的药理学。这些进展将为开发新的 治疗神经退行性疾病的药物，并更深入地了解ATP释放的功能 沟道族和差距结族。