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.

膜转运电压调节机理 SLC9膜转运蛋白家族将钠离子的导入进口质子。 它们对于调节细胞质和内体pH值至关重要，这又影响了几个 生理过程。他们的失功与许多疾病（例如糖尿病）有关 高血压，心力衰竭和癌症。特定SLC9成员的基因突变也有 与Angelman-Syndrome相关联，如疾病，多动症，家庭自闭症，癫痫和 男性不育症。 SLC9C1是SLC9家族的独特成员。与其他SLC9不同 具有膜定界钠氢交换（NHE）域的特征，通常很短 相对非结构化的C末端可溶性域，SLC9C1结合了NHE，电压感应 域（VSD）和环核苷酸结合结构域（CNBD），通过长，结构化互连 接头，在单个多肽中。最近的基础实验表明膜 循环核苷酸的超极化和结合通过SLC9C1增强离子转运。它是 独特的设计使得无法预测该蛋白的电压和配体调节 在结构层面上表现出来。 SLC9C1表现出精子特异性表达，已显示 对于小鼠和人类的精子运动至关重要。精子运动是由 膜电压，细胞内营地水平和pH的变化以及所有这些刺激的影响 SLC9C1直接介导的离子交换，使理解分子至关重要 这种不同监管的基础。为此，在此提案中，我们将整合单一的 粒子冷冻电子显微镜和生化和重建技术 探索SLC9C1关键生物物理机制的电生理方法。在AIM 1中，我们 将确定SLC9C1的第一个高分辨率结构，并确定关键交互 管理其组织。在AIM 2中，我们将阐明SLC9C1中的结构重排 由环状核苷酸结合触发并使用电生理学测试钥匙界面的作用 在介导CNBD的调节作用时。在AIM 3中，我们将确定pH和Pereant如何 离子会影响SLC9C1的结构和功能。该提案具有强大的科学基础 基于我们严格的初步研究。它具有创新性，因为它将提供第一个快照 在不同的构象中的新型膜蛋白和测试挑衅性假设 转运蛋白的电压和循环核苷酸调节的机理。获得的见解 根据我们的研究，将有助于基于结构的药物设计，以治疗男性不育症。它也会 对SLC9调节的结构和功能机制具有广泛的影响 细胞质领域进一步强调了其对人类健康的重要性。