Ion channel-transporter interactions

 描述（由申请人提供）：KCNQ 1电压门控钾（Kv）通道孔形成（a）亚基广泛表达，与危及生命的人类疾病（包括长QT综合征、房颤和糖尿病）相关。KCNQ 1通过与KCNE家族β亚基的共组装而表现出高度的功能灵活性，促进可兴奋细胞复极化的作用，并在极化细胞中作为组成性活性K+通道。 上皮细胞Na+-偶联溶质转运对于离子和溶质（包括糖和肌醇，细胞信号分子的重要渗透剂和前体）的摄取至关重要。我们最近发现KCNQ 1与几种不同的Na+偶联溶质转运蛋白形成复合物，并且这些新型复合物中至少有两种是甲状腺和脉络丛正常上皮细胞活性所需的。通过体外功能研究，我们发现， KCNQ 1和Na+依赖的肌醇转运蛋白SMIT 1和SMIT 2相互调节功能。我们最近还鉴定了其他几种通道-转运蛋白相互作用，包括KCNQ 4-SMIT 1、KCNQ 1-SGLT 1（Na+偶联葡萄糖转运蛋白）和KCNQ 1-NIS（Na+/I-同向转运蛋白）。在这里，我们将阐明这种新型的和潜在的广泛的大分子信号复合物的功能，相互作用和生理相关性的分子机制。三个主要问题得到解决。首先，哪些通道域和功能调节转运蛋白活性？使用诱变，药理学试剂和功能分析，我们将测试的假设，KCNQ 1孔和电压传感器模块可以独立地影响活动的共组装溶质转运蛋白。我们还将使用蛋白质生物化学结合通道嵌合体和诱变，以阐明与转运蛋白的物理相互作用，在体外至关重要的通道结构域。第二，为什么需要通道转运蛋白复合物？使用电生理和溶质摄取测定在体外，我们将测试的假设，KCNQ 1作为一个生物传感器与SMIT 1和SMIT 2的复合物，促进渗透压，膜脂质组成，pH值和Ca 2+的变化的反应。第三，通道转运蛋白复合物在体内的什么地方发生？在几个基因敲除小鼠品系的帮助下，我们将定位通道转运蛋白复合物，并利用正电子发射断层扫描技术来特异性地测试SGLT家族转运蛋白的KCNQ 1-KCNE调节的体内需求。Na+偶联溶质转运蛋白和Kv通道a亚基（包括KCNQ 1）具有广泛的分布、广泛的组织表达重叠和高度的生物医学意义。与我们最近的研究结果一起，这表明钾通道转运蛋白复合物有可能在哺乳动物生理学和许多流行的人类疾病的发病机制中具有高度影响力。

 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.