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(~80 mV)，在机械电转导过程中增强阳离子流入毛细胞的驱动力。EP的重要性得到了以下事实的支持：降低EP的药物是耳毒性的，而废除EP的实验操作会导致听力阈值降低或完全耳聋。我们推测，EP的产生和维持是由中间细胞(IC)和边缘细胞(MC)顶膜上的一组K+通道以及与NKCC1和Na+/K+ATPase结合的基侧Cl-通道组成的。我们进一步预测，耳蜗管K+的调节与内壁细胞(Reissner膜，RM)和内淋巴囊(ES)细胞的K+通道活动密切相关。在上一个赠款周期中，我们在实现该提案的目标方面取得了实质性进展。在下一个资助周期中，我们将重点关注：1)阐明耳蜗管(CD)内侧壁细胞(MWs和LWs)中K+通道亚型的特性和基本性质。我们将把这些基本的重要研究扩展到内淋巴囊和内淋巴管(ES/D)。2)确定耳蜗细胞特异性K+通道的分子同一性、细胞定位和密度。3)确定K+通道和Cl-通道的不同特征、通道的结合伙伴及其化学计量比、密度和表达的极性，这些特性赋予它们在CD中的独特特性以赋予EP。4)利用CD细胞胚胎学和细胞特异性基因/启动子表达的重要特征，建立细胞特异性K+通道缺失/改变的小鼠模型。这将检验一种假设，即内耳的K+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。我们将运用创新的分子生物学、电生理学和成像技术，其中许多技术来自以前的耳蜗管K+通道研究，以发现K+通道生理学的基本领域，以及内耳产生EP和K+稳态的机制。总的来说，这些研究将极大地扩展我们对EP产生的细胞机制的理解。在这些研究中，具有实用意义的是开发可用于减轻与内耳K+通道功能障碍相关的听力损失的策略的诱人可能性。 与公共健康相关：内耳有一种独特的电势，称为耳蜗内电位(&gt；80 mV)，这是正常听力所必需的。有证据表明，在大多数年龄相关性听力损失的动物模型中，听力阈值/敏感度与~1dB/mV的EP直接相关，这一证据支持了EP对正常听力的重要性。我们假设EP是由内耳壁细胞中的一支K+通道产生和维持的。我们将确定内耳壁细胞的特性。此外，我们将使用各种分子生物学、生化和功能技术来克隆和鉴定耳蜗壁特异的K+和Cl-通道。这些通道在体外的生理作用将被确定。最后，我们将通过在小鼠的STV中使用细胞特异性显性-负性(DN)策略削弱通道的功能来确定通道在体内的功能作用。总之，这些研究将极大地扩展我们对单个K+和Cl-通道的特定功能的理解，以及耳蜗壁中不同类型的细胞，以及它们如何在体内共同调节EP和跨上皮离子转运过程。

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.