Structure and function of chloride channels and transporters

描述（由申请人提供）：CLC家族的氯离子通道和转运体在许多生理过程中起着至关重要的作用，包括神经和肌肉细胞的电兴奋性调节，盐和水在上皮中的运动调节以及细胞内区室的酸化。人类基因组编码9个clc，其中5个是转运体，4个是通道。其中5个基因的突变会导致合成功能改变的蛋白质，从而导致遗传性疾病，如先天性肌强直、巴特氏综合征、登特氏病、骨质疏松症和癫痫。这些蛋白参与如此广泛的生理和病理过程，标志着它们是治疗方法和药物设计发展的理想靶点。然而，由于我们缺乏对CLC功能的基本结构和机制基础的了解，这一进展受到阻碍。这一提议旨在提供CLC蛋白如何调节跨膜Cl-通量以及这些如何与H+运动耦合的分子描述。这一目标将通过结合使用x射线晶体学、电生理记录、通量测量以及首次对CLC蛋白进行的直接底物结合测量来实现。CLC蛋白是同型二聚体，其中每个单体形成一个独立的渗透途径。所有家族成员都催化Cl-离子穿过细胞膜的运动，但可以通过两种相反的热力学机制之一来实现：CLC通道消散Cl-电化学梯度，而CLC转运体以牺牲H+电化学梯度为代价催化Cl-向上运动，反之亦然。我们的第一个主要目标是确定允许CLC转运体催化Cl-和H+跨细胞膜的化学计量交换的分子步骤。转运体经历一系列复杂的构象变化，使它们能够将储存在电化学梯度中的能量转化为上坡的基底运动。我们将在这一过程中识别、隔离和描述关键的结构性参与者。我们的第二个主要目标是确定CLC蛋白阴离子选择性的分子基础。底物特异性对于通道和转运体的正常功能至关重要。我们现在已经确定了几个对这个过程可能至关重要的残留物。为了改变CLC通道和转运体的底物特异性，我们将通过对这些残基的诱变来操纵结合和渗透性的选择性来验证这一假设。有人提出CLC转运体和通道具有共同的结构。本提案的第三个目标是通过将前者转化为后者来检验这一假设。我们将通过追求两种互补的方法来实现这一目标：首先，我们将识别并消除阻碍Cl-通过转运蛋白移动的物理障碍；其次，我们将识别区分通道和转运蛋白的关键残基，并将它们突变为其他的。公共卫生相关性：CLC通道和转运体介导阴离子跨细胞膜运输，调节肌肉和神经的电兴奋性，允许盐和水跨上皮运动，参与沿内体-溶酶体途径的囊泡酸化和神经递质释放囊泡。9个人类CLC基因中的5个发生突变可导致遗传性疾病。由于这些通道和转运体在所有这些生理过程中的关键作用，了解这些蛋白质的功能机制将对治疗有用。通过将这些蛋白质的结构与其功能联系起来，最终有可能开发或鉴定出能够增强或抑制Cl-转运的药物，这取决于靶点和最终目的。

DESCRIPTION (provided by applicant): Chloride channels and transporters of the CLC family play crucial roles in a myriad of physiological processes including regulation of electrical excitability of nerve and muscle cells, modulation of salt and water movement across epithelia and acidification of intracellular compartments. The human genome encodes for 9 CLCs, 5 of which are transporters and 4 are channels. Mutations in 5 of these genes lead to the synthesis of proteins with altered functionalities that cause genetically inherited disorders such as myotonia congenita, Bartter's syndrome, Dent's disease, osteopetrosis and epilepsy. The involvement of these proteins in such a wide array of physiological and pathological processes marks them as ideal targets for the development of therapeutic treatments and drug design. This progress is, however, stunted by our lack of knowledge of the basic structural and mechanistic underpinnings underlying CLC function. This proposal aims to provide a molecular description of how CLC proteins regulate transmembrane Cl- fluxes and how these are coupled to H+ movement. This goal will be pursued through the combined use of X-ray crystallography, electrophysiological recordings, flux measurements and, for the first time for CLC proteins, direct substrate binding measurements. CLC proteins are homodimers where each monomer forms an independent permeation pathway. All family members catalyze movement of Cl- ions across cellular membranes, but can do so via either of two thermodynamically opposing mechanisms: the CLC channels dissipate the Cl- electrochemical gradient, whereas the CLC transporters catalyze uphill Cl- movement at the expense of the H+ electrochemical gradient, or vice versa. Our first major aim is to identify the molecular steps that allow CLC transporters to catalyze the stoichiometric exchange of Cl- and H+ across cellular membranes. Transporters undergo a complex series of conformational changes that allow them to transform the energy stored in electrochemical gradients into uphill substrate movement. We will identify, isolate and characterize the crucial structural players in this process. Our second major aim is to identify the molecular basis of anionic selectivity in CLC proteins. Substrate specificity is of paramount importance to proper function of both channels and transporters. We have now identified several residues potentially crucial for this process. We will test this hypothesis by manipulating through mutagenesis of these residues the selectivity of binding and permeability in order to alter the substrate specificity of the CLC channels and transporters. It has been proposed that CLC transporters and channels share a common architecture. The third goal of this proposal is to test this hypothesis by transforming the former into the latter. We will accomplish this by pursuing two complementary approaches: first, we will identify and eliminate the physical barriers blocking Cl- movement through the transporters and, second, we will identify the key residues differentiating the channels and the transporters and mutate the ones into the others. PUBLIC HEALTH RELEVANCE: The CLC channels and transporters mediate anion transport across cellular membranes to modulate the electrical excitability of muscle and nerve, to allow salt and water movement across epithelia, participate in acidification of vesicles along the endosomal- lysosomal pathway and of neurotransmitter release vesicles. Mutations in 5 of the 9 human CLC genes lead to genetic diseases. Because of the crucial role of these channels and transporters in all of these physiological processes, understanding the mechanism of function of these proteins will be therapeutically useful. By relating the structure of these proteins to their function, it may ultimately be possible to develop or identify pharmaceutical agents that could either enhance or inhibit Cl- transport, depending on the target and the ultimate goal.