Mechanism and function of intracellular sodium-proton exchangers

摘要 越来越多的严重疾病，从综合症自闭症、智力残疾到癌症 大脑和肠道的部分被连接到一个电子中和型Na/H交换器家族的细胞内成员， 包括内体异构体NHE6和NHE9(ENHE)，调节细胞内的pH和Na 内切-溶酶体途径。质膜NHE亚型已得到彻底的表征和 药物靶向。相比之下，由于局限性和挑战，细胞内NHE的研究仍然很少 在感测细胞器特定的管腔离子方面。此外，eNHE亚型和eNHE亚型的重叠分布 关于细胞器内钠和质子运输方向的相互矛盾的报告阻碍了 对转运蛋白功能和生理学的机械理解。疾病与eNHE基因有关的病例报告 变异是零星的，而且基因-表型相关性不完全。这项提案汇集了三个 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应对 这些问题。为了克服测量这些转运体活动的技术挑战，我们有 研制了一种可同时检测Na和H的多功能荧光报告装置，用于精确测定细胞内Na/H 交换。该记者可以针对特定细胞器同时读出Na和H水平 其中使用了一种称为双离子测量的成像方法。在目标一中，我们将部署记者到具体 沿着内切溶酶体途径的间隔室，以量化健康和疾病状态下的[Na]和[H]。 我们将确定单个eNHE亚型在KEY中的功能贡献和运输方式 细胞器。这一目标将为临床上影响eNHE的基因变异的功能分析奠定基础。 为了捕捉eNHE的疾病图景，在目标2中，我们将评估罕见和 SLC9A6和SLC9A9中常见的基因变异。对于这些分析，我们将利用大规模的exome 对临床队列进行排序，并与他们未识别的电子健康记录配对。组合遗传 关联、基因表达和功能分析将提供对生物基础的机械洞察 与eNHE相关的疾病。综上所述，我们对内切溶酶体的全面生化图谱 将基因变异和表达与表型联系起来的途径和疾病不可知的方法将获得广泛的 一系列的细胞和临床相关性，将为成功治疗这些疾病铺平道路 疾病中的转运者。

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.