Signals Regulating Vestibular Endolymph Homeostasis

描述(申请人提供)：内耳对人类健康至关重要，因为它提供听力和平衡控制所需的关键感官信息。内耳的听觉部分和前庭部分是充满液体的腔室，其功能分别是检测声波和重力/加速度/运动。更多地了解调节内耳内淋巴液产生的分子，应该有助于洞察影响人类群体的听力和平衡障碍。初步研究表明，EphB2受体酪氨酸激酶突变的成年小鼠表现出与前庭功能缺陷一致的旋转运动。在相关的初步研究中，在其同源跨膜伙伴ewitin-B2中杂合突变的成年小鼠也被发现表现出旋转运动。这两个突变体都显示出前庭器官内淋巴液的产生大大减少，而前庭器官通常富含钾。对椭圆囊细胞外液的直接测量显示，在EphB2和eaffin-B2突变体中，内淋巴钾浓度和内淋巴电位显著降低。这种液体产生的缺陷与EphB2在分泌性前庭暗细胞中的表达受限以及ePhin-B2在邻近的前庭移行细胞中的表达受限相一致。暗细胞和移行细胞是膜性内耳上皮细胞的亚室，在调节内淋巴液的离子稳态方面起着重要作用。由于肾上腺素和Eph受体被认为在细胞-细胞信号转导中发挥重要作用，本申请中描述的初步数据为控制内淋巴液的产生和动态平衡的信号分子提供了新的见解。重要的是，这些数据为研究前庭功能障碍提供了两个新的动物模型系统。我们计划在特定的目标1和2中扩大这些遗传学实验，通过在EphB2和ePhin-B2基因中产生点突变来帮助确定内淋巴产生是否利用了酪氨酸激酶或PDZ信号通路。另外的初步生化研究表明，某些结合了EphB2和eaffin-B2的C-末端尾巴的PDZ结构域蛋白也能够结合其他与调节流体动态平衡有关的跨膜蛋白的C-末端尾巴，包括水通道蛋白水通道和阴离子交换器。在具体目标3中，我们计划通过关注EphB2与分子之间可能的物理联系来扩展这些生化研究，这些分子在维持内淋巴动态平衡方面具有重要作用。本文概述的遗传和生化研究的总体目标是更好地了解EphB2和ePhin-B2介导的信号如何与内淋巴液的产生和正常的前庭功能有关。

DESCRIPTION (provided by applicant): The inner ear is essential for human health as it provides key sensory information required for hearing and balance control. The auditory and vestibular components of the inner ear are fluid-filled chambers that function to detect sound waves and gravity/acceleration/movement, respectively. A greater understanding of the molecules which regulate the production of the endolymph fluid in the inner ear should provide insight into hearing and balance disorders that affect the human population. Preliminary studies show that adult mice mutant for the EphB2 receptor tyrosine kinase display a circling locomotion consistent with a defect in vestibular function. In related preliminary investigations, adult mice heterozygous for a mutation in its cognate transmembrane partner, ephrin-B2, are also found to exhibit a circling locomotion. Both mutants display a much reduced production of endolymph fluid in the vestibular apparatus, which is normally rich in potassium. Direct measurement of the extracellular fluid in the utricle reveals a highly significant decrease in endolymph potassium concentration and endolymphatic potential in the EphB2 and ephrin-B2 mutants. Such a defect in fluid production is consistent with the restricted expression of EphB2 to the secretory vestibular dark cells and ephrin-B2 to adjacent vestibular transitional cells. Dark and transitional cells are subcompartments of the membranous inner ear epithelia that play important roles in regulating the ionic homeostasis of endolymph fluid. As the ephrins and Eph receptors are thought to play important roles in cell-cell signaling, the preliminary data described in this application provides novel insight into the signaling molecules that may control the production and homeostasis of endolymph fluid. Importantly, the data provide two new animal model systems to study vestibular dysfunction. We plan to expand on these genetic experiments in Specific Aims 1 and 2 by generating point mutations in the EphB2 and ephrin-B2 genes to help determine whether tyrosine kinase or PDZ signaling pathways are utilized for endolymph production. Additional preliminary biochemical studies indicate certain PDZ domain proteins that bind the C-terminal tails of both EphB2 and ephrin-B2 are also able to bind the C-terminal tails of other membrane spanning proteins implicated in regulating fluid homeostasis, including aquaporin water channels and anion exchangers. In Specific Aim 3, we plan to expand on these biochemical studies by focusing on possible physical associations of EphB2 with molecules implicated to have important roles in maintaining endolymph homeostasis. The overall objective of the genetic and biochemical studies outlined here are to obtain a better understanding how signaling mediated by EphB2 and ephrin-B2 is linked to the production of endolymph fluid and normal vestibular function.