Mechanism and function of intracellular sodium-proton exchangers

细胞内钠质子交换器的机制和功能

• 批准号: 10797218
• 负责人: Yamuna Krishnan
• 金额: $ 9.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797218
• 关键词: Biochemical; Biological; Biological Assay; Case Study; Cell membrane; Cells; Clinical; Disease; Electronic Health Record; Endosomes; Family; Gene Expression; Genes; Genotype; Individual; Intellectual functioning disability; Ions; Link; Lysosomes; Malignant Neoplasms; Malignant neoplasm of brain; Maps; Measurement; Measures; Molecular; Mutation; Organelles; Pathway interactions; Phenotype; Physiology; Protein Isoforms; Protons; Public Health; Reporter; Reporting; Research; Resources; Sodium; Time; autism spectrum disorder; clinical phenotype; clinically significant; cohort; endosome membrane; exome sequencing; genetic association; genetic variant; imaging modality; insight; member; patient population; therapeutic target; tool

SUMMARY A growing number of serious disorders ranging from syndromic autism and intellectual disability to cancers of the brain and gut have been linked to intracellular members of a family of electroneutral Na+/H+ exchangers, including endosomal isoforms NHE6 and NHE9 (eNHE), that regulate pH and Na+ within the compartments of the endo-lysosomal pathway. Plasma membrane NHE isoforms have been thoroughly characterized and pharmaceutically targeted. In contrast, intracellular NHE remain poorly studied due to limitations and challenges in sensing organelle-specific lumenal ions. Furthermore, overlapping distributions of eNHE isoforms and contradictory reports on the direction of sodium and proton transport within organelles has hindered a mechanistic understanding of transporter function and physiology. Case reports linking disease to eNHE genetic variants are sporadic and genotype-phenotype correlations are incomplete. This proposal brings together three research groups with unique and complementary expertise, together with powerful tools and resources to tackle these problems. To overcome the technical challenges in measuring the activity of these transporters, we have developed a multi-functional fluorescent reporter for both Na+ and H+ to precisely assay intracellular Na+/H+ exchange. This reporter can be targeted to specific organelles to simultaneously read out Na+ and H+ levels therein using an imaging method called two-ion measurement. In Aim 1, we will deploy this reporter to specific compartments along the endo-lysosomal pathway to quantify [Na+] and [H+] in both healthy and disease states. We will determine the functional contribution and mode of transport of individual eNHE isoforms in key organelles. This aim will lay the groundwork for functional analysis of clinically impactful gene variants in eNHE. To capture the disease landscape for eNHE, in Aim 2 we will evaluate the clinical significance of rare and common gene variants in SLC9A6 and SLC9A9. For these analyses, we will leverage large-scale exome sequencing of a clinical cohort, paired with their de-identified electronic health records. Combining genetic associations, gene expression and functional analysis will provide mechanistic insight on the biological basis of disease associated with eNHE. In summary, our comprehensive biochemical mapping of the endo-lysosomal pathway and disease-agnostic approach to link gene variants and expression to phenotypes will capture a broad range of cellular and clinical correlates that will pave the way to successful therapeutic targeting of these transporters in disease.

概括 越来越多的严重疾病，从综合症自闭症和智力残疾到癌症 大脑和肠道的脑部与电击Na+/H+交换器家族的细胞内成员有关， 包括内体同工型NHE6和NHE9（ENHE），这些（ENHE）调节pH和Na+在 内部溶菌体途径。质膜NHE同工型已被彻底表征，并且 药物针对性。相反，由于局限性和挑战，细胞内NHE的研究仍然很差 在传感细胞器特异性腔离子时。此外，增强同工型的重叠分布和 关于细胞器中钠和质子转运方向的矛盾报告阻碍了 机械理解转运蛋白功能和生理学。疾病联系以增强遗传的病例报告 变体是零星的，基因型 - 表型相关性不完整。该提议汇集了三个 具有独特和互补专业知识的研究小组，以及强大的工具和资源来解决 这些问题。为了克服衡量这些转运蛋白活动的技术挑战，我们有 开发了针对Na+和H+的多功能荧光记者，以精确测定细胞内Na+/H+ 交换。该记者可以针对特定细胞器，以同时读取Na+和H+级别 在其中使用称为两离子测量的成像方法。在AIM 1中，我们将将此记者部署到特定 在健康和疾病状态下，沿着内部溶菌体途径进行量化[Na+]和[H+]的隔室。 我们将确定钥匙中个体增强同工型的功能贡献和运输方式 细胞器。该目标将为临床上有影响力的基因变体的功能分析奠定基础。 为了捕获疾病景观以增强疾病，在目标2中，我们将评估稀有和 SLC9A6和SLC9A9中的常见基因变体。对于这些分析，我们将利用大规模外显子 对临床队列的测序，并与其去识别的电子健康记录配对。结合遗传 关联，基因表达和功能分析将根据生物学基础提供机械洞察力 与增强有关的疾病。总而言之，我们的内部溶酶体的全面生化映射 将基因变异和表达与表型联系起来的途径和疾病敏锐的方法将捕获广泛的 细胞和临床相关的范围将为这些成功靶向的成功铺平道路 疾病转运蛋白。