Structure and function of chloride channels and transporters

描述(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发挥关键作用，包括调节神经和肌肉细胞的电兴奋性，调节跨越上皮的盐和水的移动，以及细胞内隔室的酸化。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个基因的突变导致蛋白质合成功能改变，导致遗传性疾病，如先天性肌强直、巴特综合征、登特氏病、骨质疏松症和癫痫。这些蛋白质参与了如此广泛的生理和病理过程，这标志着它们是开发治疗和药物设计的理想靶点。然而，由于我们缺乏对潜在的CLC功能的基本结构和机制基础的了解，这一进展受到阻碍。这项建议旨在提供CLC蛋白如何调节跨膜Cl-通量以及这些跨膜通量如何与H+运动相耦合的分子描述。这一目标将通过联合使用X射线结晶学、电生理记录、通量测量以及首次用于CLC蛋白质的直接底物结合测量来实现。CLC蛋白是同源二聚体，每个单体形成一个独立的渗透途径。所有家族成员都催化氯离子在细胞膜上的移动，但可以通过两种热力学相反的机制之一来实现这一点：ClC通道消散了Cl-的电化学梯度，而ClC转运体以H+的电化学梯度为代价催化了Cl-的上行移动，反之亦然。我们的第一个主要目标是确定允许CLC转运蛋白催化跨细胞膜的氯和H+的化学计量交换的分子步骤。转运蛋白经历了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行的底物运动。我们将确定、隔离和确定这一进程中关键的结构性参与者的特征。我们的第二个主要目标是确定CLC蛋白中阴离子选择性的分子基础。底物专一性对通道和转运蛋白的正常功能至关重要。我们现在已经确定了几个对这一过程可能至关重要的残留物。我们将通过突变这些残基来操作结合和通透性的选择性，以改变CLC通道和转运蛋白的底物特异性，以检验这一假设。已经提出了CLC传输器和通道共享公共体系结构。这一提议的第三个目标是通过将前者转化为后者来检验这一假设。我们将通过两个互补的方法来实现这一点：第一，我们将识别和消除阻止氯离子通过转运体移动的物理障碍，第二，我们将识别区分通道和转运体的关键残基，并将一个突变为其他。

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.