Structural and functional studies of CALHM channels

CALHM通道的结构和功能研究

• 批准号: 10155599
• 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/10155599
• 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; 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保护和神经保护作用的神经系统活动中起着基本作用， 突触传播，伤害感受，感染，听力和口味。此过程是通过发布的 三磷酸腺苷（ATP）穿过经典胞吐作用或ATP渗透 通道进入合成裂缝，ATP在相邻单元上结合下游接收器。有五个 ATP释放通道的家族：连接蛋白，泛毒素，体积调节的阴离子通道，最大通道和 钙稳态调节剂（CALHMS）。在大脑和味蕾中高度表达，CALHM通道 在味觉和神经元传播中扮演重要角色，它们的失调与各种 神经系统疾病，包括阿尔茨海默氏病，缺血性脑损伤和抑郁症，使CALHM 通道重要的药物目标。 CALHM家族由三个成员组成，分别是Calhm1、2和3。它们依赖电压，细胞外， 钙浓度调节的非选择性离子通道，可渗透到信号分子ATP 和钙。预计它们将与连接蛋白，pennexins，innexins和vracs共享膜拓扑。 功能研究提供了共识的观点，即CALHM1形成了六聚体渠道，并且仅形成 半通道，但没有间隙连接。 CALHMS活动受到多种因素的调节 Ruthenium Red，GD3+和2-APB。尽管Calhms是人类生理学的核心，并且是潜在的 治疗靶标，这个家庭没有结构。我们不理解，分子细节 通道被激活或抑制，或如何通过特定位点的小分子结合来调节。 我们已经发表了有力的证据，表明CALHM2是未定的，并且是半通道和差距 体外交界。我们已经确定了在CA2+的无开放状态下人类CalHM2的冷冻EM结构，以及 红色 - 边界抑制状态。这些初步结果不仅提供了A的第一个原子结构 CALHM家庭成员，也是抑制状态下的第一个真正的善意结构，从未报道过 对于具有类似拓扑的通道，包括连接蛋白，泛毒素，innexins和vracs。我们观察到一种结合 以前是完全未知的ruthenium红色的位置。在此初步数据的基础上，我们建议 继续对CalHM2和其他两个家庭成员CalHM1和CalHM3进行结构研究，合并 使用完整的电生理实验和其他功能方法，以定义分子 综合门控机制以及间隙连接的分子决定和功能的基础 形成及其药理学。这些进步将为开发新的基础提供坚实的基础 针对神经退行性疾病的药物，并更深入地了解ATP释放的功能 渠道家庭和缝隙连接家族。