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，耳蜗管的钾调节与内壁细胞(Reissner膜，RM)和内淋巴囊(ES)细胞的钾通道活动密切相关。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，我们将重点关注：1)阐明耳蜗管(CD)内侧壁和外侧壁(MWs和LWs)细胞中K通道亚型的身份和基本性质的未解方面。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)确定耳蜗肌、LW和ES/D细胞中细胞特异性钾通道的分子同一性、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、密度和表达的极性，这些特性赋予它们在CD中的独特特征以赋予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动态平衡的机制。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策略的诱人可能性，这些策略可以用来减轻与内耳K通道功能障碍相关的听力损失。

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.