Potassium Homeostasis in the Inner Ear

描述（由申请人提供）：我们试图了解耳蜗内电位（EP）产生的详细机制，EP是一种细胞外正电位（~ 80mv），在机电转导过程中促进阳离子流入毛细胞的驱动力。降低EP的药物具有耳毒性，而消除EP的实验操作会导致听力阈值下降或完全耳聋，这一事实支持了EP的重要性。我们假设EP是由中间细胞（ic）和边缘细胞（MCs）的顶膜上的K+通道以及与NKCC1和Na+/K+ atp酶结合的基底侧Cl-通道产生和维持的。我们进一步预测，耳蜗管内K+的调节与内侧壁（Reissner’s膜，RM）和内淋巴囊（ES）细胞中K+通道的活性密切相关。在上一个拨款周期，我们在实现建议的目标方面取得了重大进展。对于下一个拨款周期，我们将重点关注：1)澄清耳蜗导管（CD）内侧壁细胞（MWs & LWs）中K+通道亚型的身份和基本特性的未解决方面。我们将把这些基本重要的研究扩展到内淋巴囊和导管（ES/D）。2)测定耳蜗MW、LW和ES/D细胞中细胞特异性K+通道的分子特性、细胞定位和密度。3)确定K+和Cl-通道的独特特征，通道的结合伙伴及其化学计量、密度和表达极性，使其在CD中具有无与伦比的特性，从而赋予EP。4)利用CD细胞胚胎学的重要特征和基因/启动子的细胞特异性表达，生成具有细胞特异性K+通道缺失/改变的小鼠模型。这将验证内耳中K+的调节、EP的产生和维持依赖于CD的MW和LW中K+通道的细胞特异性表达的假设。我们将采用创新的分子生物学、电生理学和成像技术，其中许多技术的灵感来自于以前耳蜗管K+通道的研究，以发现基本的、K+通道生理学的新通道和内耳EP和K+稳态产生的机制。总的来说，这些研究将大大扩展我们对EP产生的细胞机制的理解。在这些研究中，具有实用重要性的是，开发可能用于减轻内耳K+通道功能障碍相关听力损失的策略的诱人可能性。

DESCRIPTION (provided by applicant): We seek to understand the detailed mechanisms for the generation of the endocochlear potential (EP), an extracellular positive potential (~80 mV) that boosts the driving force for the influx of cations into hair cells during mechanoelectrical transduction. The importance of EP is underpinned by the fact that drugs whose effects decrease EP are ototoxic and experimental manipulations that abolish EP result in a decreased hearing threshold or total deafness. We hypothesize that the EP is produced and maintained by a cadre of K+ channels in the apical membrane of intermediate cells (ICs) and marginal cells (MCs), as well as basolateral Cl- channels in conjunction with NKCC1 and Na+/K+ATPase. We further predict that K+ regulation in the cochlear duct is tightly linked to the activity of K+ channels in cells of the medial wall (Reissner's membrane, RM) and endolymphatic sac (ES). We have made substantial progress towards the objectives of the proposal in the last grant cycle. For the next grant cycle, we will focus our attention on: 1) Clarifying unresolved aspects of the identity, and elementary properties of the subtypes of K+ channels, in cells of medial and lateral walls (MWs & LWs) of the cochlear duct (CD). We will extend these fundamentally important studies to the endolymphatic sac and duct (ES/D). 2) Determining the molecular identity, cellular localization, and density of cell-specific K+ channels in cells of cochlear MW, LW and ES/D. 3) Identifying distinct features of K+ and Cl- channels, binding partners of the channels and their stoichiometry, their density, and polarity of expression that endow their unequaled traits in the CD to confer EP. 4) Exploiting important features of the embryology of cells of the CD and cell-specific expression of genes/promoters to generate mouse models with cell-specific deletions/alterations of K+ channels. This will test the hypothesis that K+ regulation, EP generation, and maintenance in the inner ear is dependent on cell-specific expression of K+ channels in the MW and LW of the CD. We will deploy innovative molecular biological, electrophysiological, and imaging techniques, many inspired from previous cochlear duct K+ channel studies, to the discovery of fundamental, newly accessible arenas of K+ channel physiology and the mechanisms for the generation of the EP and K+ homeostasis in the inner ear. Collectively, these studies will substantially expand our understanding of the cellular mechanisms for the generation of EP. Of pragmatic importance in these studies is the tantalizing possibility of developing strategies that may be used to alleviate hearing loss associated with K+ channel malfunction in the inner ear. PUBLIC HEALTH RELEVANCE: The inner ear has a distinct electrical potential called the endocochlear potential (EP) (> 80 mV), which is a requisite for normal hearing. The significance of EP to normal hearing is underpinned by the evidence that in most animal models of age-related hearing loss, the hearing threshold/sensitivity is directly related to EP at ~1 dB/mV. We hypothesize that EP is produced and maintained by a cadre of K+ channels in cells of the walls of the inner ear. We will determine the properties of cells of the walls of the inner ear. Additionally, we will clone and identify cochlear wall-specific K+ and Cl- channels using a variety of molecular biological, biochemical, and functional techniques. The physiological roles of the channels in vitro will be determined. Last, we will identify the functional role of the channels in vivo by crippling the functions of the channels using cell-specific dominant-negative (DN) strategies in the StV in mice. Collectively, these studies will substantially expand our understanding of the specific functions of individual K+ and Cl- channels, as well as the different cell types in the walls of the cochlea, and how they work together to mediate EP and trans-epithelial ion transport processes in vivo.