Mechanisms of voltage regulation of membrane transport

膜运输的电压调节机制

• 批准号: 10595025
• 负责人: Sandipan Chowdhury
• 金额: $ 34.33万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595025
• 关键词: Action Potentials; Activities of Daily Living; Address; Affect; Angelman Syndrome; Architecture; Attention deficit hyperactivity disorder; Binding; Biochemical; Biological Assay; Biophysical Process; C-terminal; CCL21 gene; Cardiac; Cations; Cell Proliferation; Cells; Charge; Chemicals; Choline; Complement; Couples; Cryoelectron Microscopy; Cues; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Cytoplasm; Cytoplasmic Tail; DNA Sequence Alteration; Data; Dependence; Detergents; Development; Diabetes Mellitus; Digestive System Disorders; Disease; Drug Design; Electrophysiology (science); Endosomes; Enzymes; Epilepsy; Exhibits; Family; Family member; Fluorescence; Foundations; Functional disorder; Health; Heart failure; Homeostasis; Human; Hydrogen; Hypertension; Intervention; Investigation; Ion Channel Gating; Ion Exchange; Ion Transport; Ions; Length; Ligands; Link; Liposomes; Male Infertility; Malignant Neoplasms; Maps; Measures; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Micelles; Modeling; Molecular; Molecular Conformation; Movement; Mus; Muscular Dystrophies; Orthologous Gene; Pathologic; Peptide Hydrolases; Phosphoric Monoester Hydrolases; Photobleaching; Physiological Processes; Play; Point Mutation; Protein Family; Proteins; Protons; Regulation; Reperfusion Injury; Resolution; Role; Sea Urchins; Sodium; Sodium Chloride; Sperm Capacitation; Sperm Motility; Stimulus; Structure; System; Techniques; Testing; Thermodynamics; Transmembrane Transport; Variant; autism spectrum disorder; density; design; dimer; electric field; experimental study; innovation; insight; interfacial; member; mutant; novel; particle; pharmacologic; polypeptide; reconstitution; reconstruction; single molecule; sodium ion; sperm cell; therapy design; voltage

Mechanisms of voltage regulation of membrane transport SLC9 family of membrane transporters couple the import of sodium ions to export of protons. They are vital for regulation of cytoplasmic and endosomal pH, which in turn affect several physiological processes. Their disfunction has been linked to many diseases such as diabetes, hypertension, heart failure and cancer. Genetic mutations in specific SLC9 members have also been associated with Angelman-syndrome like disorders, ADHD, familial autism, epilepsies and male infertility. The SLC9C1 is a unique member of the SLC9 family. Unlike other SLC9s which feature a membrane delimited sodium-hydrogen exchange (NHE) domain and a usually short and relatively unstructured C-terminal soluble domain, SLC9C1 combines an NHE, a voltage-sensing domain (VSD) and a cyclic nucleotide binding domain (CNBD), interconnected via long, structured linkers, in a single polypeptide. Recent foundational experiments have revealed that membrane hyperpolarization and binding of cyclic nucleotides potentiates ion transport via SLC9C1. Its unique design makes it impossible to predict how voltage and ligand regulation of this protein is manifested at a structural level. SLC9C1 exhibits sperm-specific expression and has been shown to be critical for sperm motility in mouse and humans. Sperm motility is robustly modulated by changes in membrane voltage, intracellular cAMP levels and pH and all these stimuli influence SLC9C1 mediated ion exchange directly, making it vital to understand the molecular underpinnings of such diverse regulation. To this end, in this proposal we will integrate single- particle cryo-electron microscopy and reconstruction techniques with biochemical and electrophysiological methods to explore key biophysical mechanisms of SLC9C1. In Aim 1, we will determine the first high-resolution structure of SLC9C1 and identify the key interactions governing its organization. In Aim 2, we will elucidate the structural rearrangements in SLC9C1 triggered by cyclic nucleotide binding and use electrophysiology to test the role of a key interface in mediating the regulatory effects of the CNBD. In Aim 3, we will determine how pH and permeant ions affect the structure and function of SLC9C1. The proposal has a strong scientific foundation built on our rigorous preliminary studies. It is innovative as it will provide the first snapshots of a novel membrane protein in different conformations and test provocative hypotheses on the mechanisms of voltage and cyclic nucleotide regulation of a transporter. The insights obtained from our studies will aid structure-based drug design for treatment of male infertility. It will also have broad implications on the structural and functional mechanisms of SLC9 regulation by their cytoplasmic domains further underscoring its importance for human health.

膜运输的电压调节机制 SLC9 膜转运蛋白家族将钠离子的输入与质子的输出耦合起来。 它们对于细胞质和内体 pH 值的调节至关重要，进而影响多种 生理过程。它们的功能障碍与许多疾病有关，例如糖尿病、 高血压、心力衰竭和癌症。特定 SLC9 成员的基因突变也已 与安格曼综合症、注意力缺陷多动症、家族性自闭症、癫痫和 男性不育症。 SLC9C1 是 SLC9 系列中的独特成员。与其他 SLC9 不同的是 具有膜分隔的钠氢交换（NHE）结构域和通常短且 相对非结构化的 C 端可溶性结构域，SLC9C1 结合了 NHE（电压传感） 结构域（VSD）和环核苷酸结合结构域（CNBD），通过长的、结构化的结构相互连接 连接子，在单个多肽中。最近的基础实验表明，膜 环核苷酸的超极化和结合增强了通过 SLC9C1 的离子传输。它是 独特的设计使得无法预测该蛋白质的电压和配体调节方式 表现在结构层面。 SLC9C1 表现出精子特异性表达并已被证明 对小鼠和人类的精子活力至关重要。精子活力受到强有力的调节 膜电压、细胞内 cAMP 水平和 pH 的变化以及所有这些刺激的影响 SLC9C1 直接介导离子交换，因此了解分子交换至关重要 这种多样化监管的基础。为此，在本提案中，我们将整合单一 粒子冷冻电子显微镜和生物化学重建技术 电生理方法探索 SLC9C1 的关键生物物理机制。在目标 1 中，我们 将确定 SLC9C1 的第一个高分辨率结构并确定关键相互作用 管理其组织。在目标 2 中，我们将阐明 SLC9C1 中的结构重排 由环核苷酸结合触发并使用电生理学来测试关键界面的作用 调解 CNBD 的监管作用。在目标 3 中，我们将确定 pH 值和渗透性 离子影响 SLC9C1 的结构和功能。该提案具有坚实的科学基础 建立在我们严格的初步研究的基础上。它是创新的，因为它将提供第一个快照 不同构象的新型膜蛋白并测试有关的挑衅性假设 转运蛋白的电压和环核苷酸调节机制。获得的见解 我们的研究将有助于基于结构的药物设计，用于治疗男性不育症。它还将 对 SLC9 调控的结构和功能机制具有广泛的影响 细胞质结构域进一步强调了其对人类健康的重要性。