GATING OF CFTR CL CHANNELS BY ATP HYDROLYSIS

描述：CFTR(囊性纤维化跨膜电导调节器)是一种 氯离子通道，在分泌和吸收中起关键作用 水和电解质遍布上皮细胞。由于CFTR通道也表示 在心肌细胞中，被发现可以缩短动作电位时程和 诱导重复活动，它们被认为是致心律失常的。独一无二的 作为离子通道的CFTR通道的特点是，ATP的自由能 水解的收获是为了驱动构象的变化，这些变化是开放和关闭的 频道。利用突变的cftr和各种三磷酸腺苷或磷酸类似物的研究 已经提出了一个模型，即在两个核苷酸结合位点上的ATP水解是 紧耦合到通道孔的打开和关闭。我们的 然而，对偶联机制的分子基础的理解， 仍然是原始的。悬而未决的问题包括：什么是化学计量比 从ATP结合/水解到门控转换？中国的生化状态如何？ 在浇注过程中，ATP水解循环转变为开启和关闭状态 过渡？蛋白质的哪一部分形成了水孔？什么是 门和毛孔之间的关系？这些都是根本问题 这引起了生理学家的广泛兴趣。 正在采用一种组合的方法来解决分子生理学问题 Cftr氯通道。膜片钳技术的不同构型 将被用来记录cftr通道活动，以便细胞质和 通道阻滞剂可接近通道的胞外侧， 修改器，或通道开通器。蛋白质关键区域的突变 将致力于研究单一氨基酸的功能后果 取代了浇注和渗透/阻挡。依赖状态的化学物质 工程半胱氨酸的修饰使我们能够探索动态蛋白质 门控转换过程中的构象变化。《公约》的具体目标 项目是：目标1.了解ATP水解酶是如何与开场相耦合的 和CFTR的闭合跃迁。目的2.用PERMENT探查CFTR孔 和阴离子。目的3、研究结构与功能的关系 在CFTR的大门和孔洞之间。对分子的清楚理解 CFTR的作用机制将有助于药物的设计 用于囊性纤维化、分泌性腹泻和心脏病的治疗干预 心律不齐。

DESCRIPTION: CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is a chloride channel that plays a critical role in secretion and absorption of water and electrolytes across epithelia. Since CFTR channels are also expressed in cardiac myocytes and are found to shorten action potential duration and induce repetitive activity, they are implicated to be arrythmogenic. One unique feature of the CFTR channel as an ion channel is that the free energy of ATP hydrolysis is harvested to drive the conformational changes that open and close the channel. Studies using mutant CFTR and various ATP or phosphate analogs have suggested a model that ATP hydrolysis at two nucleotide binding sites is tightly coupled to the opening and closing of the channel pore. Our understanding of the molecular basis of the coupling mechanism, however, remains primitive. Unresolved questions include: What is the stoichiometry of ATP binding/hydrolysis to gating transitions? How are the biochemical states in ATP hydrolysis cycles translated to the open and closed states in the gating transitions? Which part of the protein forms the aqueous pore? What is the relationship between the gate and the pore? These are fundamental questions that interest a broad spectrum of physiologists. A combinational approach is being adopted to tackle the molecular physiology of CFTR chloride channels. Different configurations of the patch-clamp techniques will be used to record CFTR channel activity so that both the cytoplasmic and the extracellular side of the channel are accessible to channel blockers, modifiers, or channel openers. Mutations in critical regions of the protein will be made to study the functional consequences of single amino acid substitutions on gating and permeation/blocking. State-dependent chemical modifications of engineered cysteines allow us to explore the dynamic protein conformational changes during gating transitions. The specific aims of the project are: Aim 1. To understand how ATP hydrolysis is coupled to the opening and closing transitions of CFTR. Aim 2. To probe the CFTR pore with permeant and impermeant anions. Aim 3, To study the structure-function relationship between the gate and the pore of CFTR. A clear understanding of the molecular mechanisms of CFTR function will aid in the design of pharmacological agents for therapeutic intervention in cystic fibrosis, secretory diarrhea and cardiac arrythmia.