Structural and functional studies of CALHM channels

CALHM通道的结构和功能研究

• 批准号: 10573257
• 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/10573257
• 关键词: Adenosine Triphosphate; Alzheimer' s Disease; Anions; Architecture; Behavior; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Brain; Calcium; Cells; Chemosensitization; Complex; Connexins; Consensus; Cryoelectron Microscopy; Data; Dependence; Detergents; Development; Dyes; Electrophysiology (science); Environment; Exocytosis; Family; Family member; Foundations; GD3 Binding; 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; Nervous System Physiology; Neurodegenerative Disorders; 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; neurotransmission; novel therapeutics; particle; patch clamp; pharmacologic; preservation; receptor; sensor; small molecule; stoichiometry; therapeutic development; therapeutic target; 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.

项目概要 嘌呤能信号在神经系统的各种活动中发挥着重要作用，如神经保护、 突触传递、伤害感受、炎症、听力和味觉。这个过程是通过释放来启动的 三磷酸腺苷 (ATP) 通过经典的胞吐作用或 ATP 渗透性跨膜 通道进入突触间隙，ATP 在此与相邻细胞上的下游受体结合。有五个 ATP 释放通道家族：连接蛋白、pannexins、容量调节阴离子通道、maxi 通道和 钙稳态调节剂（CALHM）。在大脑和味蕾、CALHM 通道中高度表达 在味觉和神经元传递中发挥重要作用，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 通道重要的药理靶点。 CALHM 家族由 CALHM1、2 和 3 三个成员组成。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。预计它们与连接蛋白、pennexins、innexins 和 VRAC 共享膜拓扑结构。 功能研究提供了一个共识，即 CALHM1 形成六聚体通道，并且它仅形成 半通道，但不是间隙连接。 CALHMs 活性受多种因素调节，包括 钌红、Gd3+ 和 2-APB。尽管 CALHM 是人类生理学的核心并且具有潜在的潜力 治疗靶点，该家族没有结构。我们不明白，在分子细节上， 通道被激活或抑制，或者如何通过结合在特定位点的小分子对其进行调节。 我们发表了强有力的证据表明 CALHM2 是十一聚体并且以半通道和间隙形式存在 体外连接。我们已经确定了人类 CALHM2 在无 Ca2+ 开放状态下的冷冻电镜结构，并且 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 CALHM家族成员，也是第一个真正处于抑制状态的结构，但从未被报道过 适用于具有相似拓扑的通道，包括连接蛋白、pannexins、innexins 和 VRAC。我们观察到一个绑定 以前完全不为人知的钌红位点。基于这些初步数据，我们建议 继续 CALHM2 和其他两个家族成员 CALHM1 和 CALHM3 的结构研究 通过补充电生理学实验和其他功能方法，来定义分子 综合门控机制以及间隙连接的分子决定因素和功能的基础 的形成及其药理学。这些进步将为开发新产品奠定坚实的基础 对抗神经退行性疾病的药物以及更深入地了解 ATP 释放的功能 通道家族和间隙连接家族。