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、智力障碍到癌症 大脑和肠道的功能与电中性 Na /H 交换器家族的细胞内成员有关， 包括内体亚型 NHE6 和 NHE9 (eNHE)，它们调节细胞内的 pH 和 Na 内溶酶体途径。质膜 NHE 亚型已被彻底表征并 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 感测细胞器特异性的腔内离子。此外，eNHE 亚型的重叠分布和 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。将疾病与 eNHE 遗传联系起来的病例报告 变异是零星的，基因型-表型相关性不完整。该提案汇集了三项 研究小组拥有独特和互补的专业知识，以及强大的工具和资源来解决 这些问题。为了克服测量这些转运蛋白活动的技术挑战，我们 开发了 Na 和 H 的多功能荧光报告基因，可精确测定细胞内的 Na /H 交换。该报告器可以针对特定细胞器同时读出 Na 和 H 水平 其中使用称为双离子测量的成像方法。在目标 1 中，我们将部署该记者到特定的地点 沿着内溶酶体途径的隔室来量化健康和疾病状态下的 [Na ] 和 [H ]。 我们将确定关键 eNHE 亚型的功能贡献和运输方式 细胞器。这一目标将为 eNHE 中具有临床影响的基因变异的功能分析奠定基础。 为了了解 eNHE 的疾病状况，在目标 2 中，我们将评估罕见和罕见疾病的临床意义 SLC9A6 和 SLC9A9 中常见的基因变异。对于这些分析，我们将利用大规模外显子组 对临床队列进行测序，并与其去识别化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提供有关生物学基础的机制见解 与 eNHE 相关的疾病。总之，我们对内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捕获广泛的信息 一系列细胞和临床相关性将为成功治疗这些疾病铺平道路 疾病中的转运蛋白。

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.