Ion channel-transporter interactions

 描述(申请人提供)：KCNQ1电压门控钾(Kv)通道孔道形成(A)亚单位普遍表达，并与威胁生命的人类疾病有关，包括长QT综合征、房颤和糖尿病。KCNQ1通过与KCNE家族β亚基的共同组装显示出高度的功能灵活性，既促进了可兴奋细胞复极化的作用，也促进了极化过程中结构性活性K通道的作用 上皮细胞。Na偶联的溶质转运对于包括糖和肌醇在内的离子和溶质的吸收是至关重要的，肌醇是细胞信号分子的重要渗透剂和前体。我们最近发现KCNQ1与几种不同的钠偶联溶质转运体形成复合体，并且这些新的复合体中至少有两种是正常的上皮细胞活动所必需的--在甲状腺和脉络丛中。使用体外功能研究，我们发现 KCNQ1和钠依赖的肌醇转运体SMIT1和SMIT2相互调节彼此的功能。我们最近还发现了其他几种通道-转运体相互作用，包括KCNQ4-SMIT1，KCNQ1-SGLT1(钠偶联葡萄糖转运体)和KCNQ1-NIS(钠/i-转运体)。在这里，我们将阐明这类新的、可能广泛存在的大分子信号复合体的功能、相互作用和生理相关性的分子机制。主要解决了三个问题。首先，哪些通道结构域和功能调节转运蛋白的活动？利用诱变、药理制剂和功能分析，我们将检验KCNQ1孔和电压传感器模块可以独立影响共组装溶质转运体活性的假设。我们还将使用蛋白质生物化学结合通道嵌合体和突变来阐明通道结构域，这些结构域对于体外与转运蛋白的物理相互作用至关重要。第二，为什么需要通道-转运体复合体？利用电生理和体外溶质摄取试验，我们将检验KCNQ1在与SMIT1和SMIT2的复合体中作为生物传感器的假设，促进对渗透压、膜脂组成、pH和钙的变化的反应。第三，通道-转运体复合体在体内发生在哪里？借助于几个基因敲除的小鼠系，我们将定位通道转运蛋白复合体，并利用正电子发射断层扫描技术在体内特异性地检测SGLT家族转运蛋白对KCNQ1-KCNE调节的要求。Na偶联的溶质转运体和包括KCNQ1在内的Kv通道a亚基具有广泛的分布、广泛的组织表达重叠和很高的生物医学意义。结合我们最近的发现，这表明钾通道-转运体复合体在哺乳动物生理学和一些普遍存在的人类疾病的发病机制中具有潜在的高度影响。

 DESCRIPTION (provided by applicant): The KCNQ1 voltage-gated potassium (Kv) channel pore-forming (a) subunit is ubiquitously expressed and linked to life-threatening human disorders including Long QT syndrome, atrial fibrillation and diabetes. KCNQ1 exhibits a high degree of functional flexibility enabled by co-assembly with KCNE family β subunits, facilitating roles both in excitable cell repolarization, and as a constitutively active K+ channel in polarized epithelial cells. Na+-coupled solute transport is crucial for uptake of ions and solutes including sugars and myo-inositol, an important osmolyte and precursor for cell signaling molecules. We recently discovered that KCNQ1 forms complexes with several different Na+-coupled solute transporters, and that at least two of these novel complexes are required for normal epithelial cell activity - in the thyroid and choroid plexus. Using in vitro functional studies, we found that KCNQ1 and the Na+-dependent myo-inositol transporters SMIT1 and SMIT2 reciprocally regulate each other's function. We also recently identified several other channel-transporter interactions, including KCNQ4-SMIT1, KCNQ1-SGLT1 (Na+-coupled glucose transporter), and KCNQ1-NIS (Na+/I- symporter). Here, we will elucidate molecular mechanisms of function, interaction, and physiological relevance of this novel and potentially widespread class of macromolecular signaling complexes. Three main questions are addressed. First, which channel domains and functions regulate transporter activity? Using mutagenesis, pharmacological agents and functional analyses, we will test the hypothesis that the KCNQ1 pore and voltage sensor modules can independently influence activity of co-assembled solute transporters. We will also use protein biochemistry in conjunction with channel chimeras and mutagenesis to elucidate channel domains crucial for physical interaction with transporters, in vitro. Second, why are channel-transporter complexes required? Using electrophysiological and solute uptake assays in vitro we will test the hypothesis that KCNQ1 acts as a biosensor in complexes with SMIT1 and SMIT2, facilitating responses to changes in osmolarity, membrane lipid composition, pH and Ca2+. Third, where do channel-transporter complexes occur in vivo? Aided by several knockout mouse lines, we will locate channel-transporter complexes, and utilize positron emission tomography to specifically test for the requirement of KCNQ1-KCNE regulation of SGLT family transporters, in vivo. Na+-coupled solute transporters, and Kv channel a subunits including KCNQ1, exhibit broad distribution, wide tissue expression overlap, and high biomedical significance. Together with our recent findings, this suggests that potassium channel-transporter complexes have the potential to be highly influential in mammalian physiology and in the pathogenesis of a number of prevalent human disorders.