Signals Regulating Vestibular Endolymph Homeostasis

描述（由申请人提供）：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。内耳的听觉和前庭组件是充满液体的腔室，其功能分别是检测声波和重力/加速度/运动。对调节内耳内淋巴液产生的分子有了更深入的了解，就可以深入了解影响人类的听力和平衡障碍。初步研究表明，EphB 2受体酪氨酸激酶的成年小鼠突变体显示出与前庭功能缺陷一致的盘旋运动。在相关的初步研究中，成年小鼠杂合子突变在其同源跨膜伴侣，肝配蛋白-B2，也被发现表现出一个盘旋运动。这两种突变体都显示出前庭器内淋巴液的产生大大减少，而前庭器内淋巴液通常富含钾。胞囊中细胞外液的直接测量揭示了EphB 2和肝配蛋白-B2突变体中内淋巴钾浓度和内淋巴电位的高度显著降低。这种液体产生的缺陷与EphB 2向分泌性前庭暗细胞和ephrin-B2向相邻前庭移行细胞的受限表达一致。暗细胞和移行细胞是膜性内耳上皮细胞的亚区室，在调节内淋巴液离子稳态中起重要作用。由于肝配蛋白和Eph受体被认为在细胞-细胞信号传导中起重要作用，因此本申请中描述的初步数据提供了对可以控制内淋巴液的产生和稳态的信号传导分子的新见解。重要的是，这些数据提供了两种新的动物模型系统来研究前庭功能障碍。我们计划通过在EphB 2和ephrin-B2基因中产生点突变来扩展特定目标1和2中的这些遗传实验，以帮助确定酪氨酸激酶或PDZ信号通路是否用于内淋巴生产。另外的初步生物化学研究表明，结合EphB 2和肝配蛋白-B2两者的C-末端尾部的某些PDZ结构域蛋白也能够结合涉及调节流体稳态的其他跨膜蛋白的C-末端尾部，包括水通道蛋白水通道和阴离子交换剂。在具体目标3中，我们计划通过关注EphB 2与在维持内淋巴稳态中具有重要作用的分子的可能的物理关联来扩展这些生化研究。这里概述的遗传和生物化学研究的总体目标是更好地理解EphB 2和ephrin-B2介导的信号传导如何与内淋巴液的产生和正常前庭功能相关联。

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.