Molecular mechanism of Na+ -coupled HCO3- transporters: transport of CO3= and CO2

Na耦合HCO3-转运蛋白的分子机制：CO3=和CO2的转运

• 批准号: 10398247
• 负责人: Walter F Boron
• 金额: $ 66.06万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-27 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10398247
• 关键词: 3-Dimensional; Acid-Base Equilibrium; Acids; Active Biological Transport; Address; Anions; Bicarbonates; Binding; Binding Sites; Biological Assay; Carbon; Carbon Dioxide; Carbonates; Cell membrane; Cells; Charge; Chemistry; Computing Methodologies; Consumption; Coupled; Coupling; Data; Dental; Diffuse; Diffusion; Disease; Electrophysiology (science); Epilepsy; Equilibrium; Evolution; Extracellular Fluid; Family; Family member; Feeling suicidal; Gases; Generations; Genes; Genetic Polymorphism; Homeostasis; Human; Hypertension; Illinois; Interdisciplinary Study; Ions; Kidney; Knockout Mice; Laboratories; Lipids; Liquid substance; Maps; Measurement; Membrane; Migraine; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Mus; Mutation; Nephrons; Nucleotides; Oocytes; Organ; Pathogenesis; Pathway interactions; Physiological; Physiology; Play; Process; Proximal Renal Tubular Acidosis; Reaction; Regulation; Research; Role; Single Nucleotide Polymorphism; Site; Speed; Structure; Surface; Techniques; Testing; Thinking; Time; Transport Process; Universities; Work; autism spectrum disorder; base; extracellular; insight; interdisciplinary approach; malignant breast neoplasm; mathematical model; member; molecular dynamics; movement analysis; novel; preservation; simulation; sodium carbonate; tool; voltage clamp

− transporters (NCBTs, members of the SLC4 family) play critical roles in transepithelial HCO−3 Na+-coupled HCO3 transport, whole-body pH regulation, and intracellular pH (pHi) regulation. In this Multi-PI R01, the team will exploit powerful techniques, many developed in their respective laboratories, to elucidate molecular transport mechanisms of NCBTs, which are especially important in the kidney. These tools include surface pH (pHS) measurements, out-of-equilibrium (OOE) CO2/HCO3− solutions, macroscopic mathematical modeling (MMM) of acid-base transport in a single cell, and state-of-the-art molecular dynamics (MD) simulations of interactions between the substrates and the transport molecule, or of CO2 conduction through the NCBTs. In a multidisciplinary approach, the team will answer 2 major questions. Aim 1: Do all NCBTs carry some form of – ion pair—whereas other SLC4 “HCO3− ” transporters actually carry =—arriving or departing as the NaCO3 CO3 HCO3− per se? By monitoring pHS in voltage-clamped oocytes the team will test whether SLC4 family members − . They will address the same question in perfused proximal tubules (PTs) from wild- = ” vs. HCO3 transport “CO3 – binding to the KKMIK region type (WT) and NBCe1-A/D knockout mice. They also test the hypothesis that NaCO3 of TM5 is a rate-limiting step for NBCe1-A transport. Using MD, the team will identify/model outward-facing, occluded, and inward-facing conformational states of NBCe1, NBCn1 and AE1, and identify potential interaction sites –, =, −, Cl−, for NaCO3 Na+, CO3 HCO3 and and use MMM (3D reaction-diffusion simulations) to assess physiological data. Finally, in an iterative process, the team will assess single nucleotide polymorphisms (m- = ” vs. HCO−3 SNPs) as well as other mutations suggested by MD studies, prioritize them, and evaluate for “CO3 transport using physiological assay, interpret using MD and MMM, and suggest new mutations. Aim 2: Do all = ” transport, NCBTs conduct CO2 whereas other SLC4 transporters do not? Having presumably committed to “CO3 evolution faced the challenge of translocating the second carbon atom, ultimately derived from 2×HCO3−. The team will use electrophysiological techniques and a novel neutral buoyancy assay (NBA) to ask whether all NCBTs conduct CO2, whereas other SLC4 members do not. In perfused PTs from WT and NBCe1-A/D knockout mice, they will ask if NBCe1-A conducts CO2 in PTs. The team will use MD to identify potential CO2 pathways through NBCe1, NBCn1, and AE1 as a negative control. MMM will assess the physiological data. Finally, in an iterative process, the team will process m-SNPs and other mutations as outlined in Aim 1, but now for effects on CO2 conduction. The research will reorganize our thinking of NCBT function, providing valuable insight into the pathogenesis of proximal renal tubular acidosis (pRTA) and other maladies associated with NBCe1 (e.g., migraine, ocular and dental abnormalities, suicidal ideation), other NCBTs (e.g., hypertension, breast cancer, epilepsy, autism). The systematic analysis of m-SNPs may provide insights into previously unrecognized “NCBT- opathies.” The work also will have broader impact by elucidating physiological acid-base surface chemistry and = ”. − vs “CO3 for the first time permitting on to distinguish unambiguously among the transport of H+ vs HCO3

− 转运蛋白（NCBT，SLC4 家族成员）在跨上皮 HCO−3 中发挥关键作用 Na+偶联的HCO3 在这个 Multi-PI R01 中，该团队将研究运输、全身 pH 调节和细胞内 pH (pHi) 调节。 利用强大的技术（许多技术是在各自的实验室开发的）来阐明分子运输 NCBT 的机制，在肾脏中尤其重要，这些工具包括表面 pH 值 (pHS)。 测量、非平衡 (OOE) CO2/HCO3− 解决方案、宏观数学建模 (MMM) 单细胞中的酸碱传输以及最先进的相互作用分子动力学 (MD) 模拟 底物和运输分子之间，或 CO2 通过 NCBT 传导。 多学科方法，团队将回答 2 个主要问题 目标 1：所有 NCBT 是否都携带某种形式的内容。 – 离子对——而其他 SLC4“HCO3− ”转运蛋白实际上携带 =—以 NaCO3 形式到达或离开 二氧化碳 HCO3− 本身？通过监测电压钳制卵母细胞中的 pHS，团队将测试 SLC4 家族成员是否存在 - 他们将在野生近端小管（PT）灌注中解决同样的问题 = ” 与 HCO3 运输“CO3 – 绑定到 KKMIK 区域 他们还测试了 NaCO3 型（WT）和 NBCe1-A/D 敲除小鼠的假设。 TM5 是 NBCe1-A 传输的速率限制步骤 使用 MD，团队将识别/建模面向外的、 NBCe1、NBCn1 和 AE1 的闭塞和向内构象状态，并确定潜在的相互作用 位点 –, =, −, Cl−, 对于 NaCO3 Na+、CO3 HCO3 并使用 MMM（3D 反应扩散模拟）来评估 最后，在迭代过程中，团队将评估单核苷酸多态性（m-） = ” 与 HCO−3 SNP）以及 MD 研究建议的其他突变，对它们进行优先排序，并评估“CO3 使用生理测定进行运输，使用 MD 和 MMM 进行解释，并提出新的突变。 目标 2：完成所有操作。 =”运输， NCBT 运输 CO2，而其他 SLC4 运输商则不运输 CO3？ 进化面临着第二个碳原子易位的挑战，该碳原子最终源自 2×HCO3−。 团队将使用电生理技术和新型中性浮力测定（NBA）来询问所有测定是否 NCBT 传导 CO2，而其他 SLC4 成员在 WT 和 NBCe1-A/D 敲除的灌注 PT 中则不传导 CO2。 小鼠，他们会询问 NBCe1-A 是否在 PT 中传导 CO2。该团队将使用 MD 来识别潜在的 CO2 途径。 最后，通过 NBCe1、NBCn1 和 AE1 作为阴性对照来评估生理数据。 在迭代过程中，团队将按照目标 1 中所述处理 m-SNP 和其他突变，但现在要考虑对 该研究将重新组织我们对 NCBT 功能的思考，为我们提供有价值的见解。 近端肾小管酸中毒 (pRTA) 和其他与 NBCe1 相关的疾病（例如， 偏头痛、眼部和牙齿异常、自杀意念）、其他 NCBT（例如高血压、乳腺癌、 m-SNP 的系统分析可能为以前未被认识的“NCBT-”提供见解。 这项工作还将通过阐明生理酸碱表面化学和 =”。 − 与“CO3 首次允许明确地区分 H+ 与 HCO3 的运输