Mechanism and function of intracellular sodium-proton exchangers

总结 越来越多的严重疾病，从综合征自闭症和智力残疾到癌症， 脑和肠道的神经元与电中性Na+/H+交换器家族的细胞内成员有关， 包括内体亚型NHE 6和NHE 9（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， 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 在感应细胞器特定的内腔离子。此外，eNHE亚型和 关于钠和质子在细胞器内转运方向的相互矛盾的报道阻碍了 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 变异是散发的，基因型-表型相关性是不完全的。该提案汇集了三个 拥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，以解决 这些问题为了克服测量这些转运体活动的技术挑战，我们 开发了Na+和H+的多功能荧光报告分子，以精确测定细胞内Na+/H+ 交易所该报告子可靶向特定细胞器，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，我们将把这名记者部署到具体的 沿着内-溶酶体途径的沿着区室来定量健康和疾病状态中的[Na+]和[H+]。 我们将确定关键的单个eNHE亚型的功能贡献和转运模式， 细胞器这一目标将为eNHE中具有临床影响力的基因变体的功能分析奠定基础。 为了捕捉eNHE的疾病景观，在目标2中，我们将评估罕见和 SLC 9A 6和SLC 9A 9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组 临床队列的测序，与他们的去识别电子健康记录配对。结合遗传 协会，基因表达和功能分析将提供机制的洞察力的生物学基础上， 与ENHE相关的疾病。总之，我们对内溶酶体的全面生化作图 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 一系列的细胞和临床相关性，这将铺平道路，成功的治疗靶向这些 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.