Atomic basis for chloride channel and transporter gating and selectivity

摘要 CLC通道和转运体通过生物膜调节阴离子的运输。编码基因 因为CLC蛋白存在于几乎所有的生物体中，从细菌到植物和人类。基因突变改变了 编码CLC同源物的九个人类基因中的五个的特性导致了骨骼的遗传疾病， 肾脏、大脑和肌肉，强调这些蛋白质在各种组织和 细胞隔间。尽管它们在病理生理上很重要，但我们对这些蛋白质如何 功能远远落后于许多其他类别的离子通道和交换器。这限制了我们的能力 解释它们在人体生理学中的作用，并设计有针对性的药物干预措施 有选择地操纵他们的活动。因此，我们的长期目标是阐明ClC Cl-的原子基础。 通道和传输器功能。我们的建议有三个具体目标，每个目标都涉及 关于《中图法》功能的基本悬而未决的机制问题。我们对协同效应的创新使用 实验和计算方法使具体假设的表述及其严谨 测试。在第一个目标中，我们将确定底物选择性在ClC氯离子通道中的基础。而当 阳离子通道的选择性是众所周知的，但对阴离子的选择性几乎一无所知。我们将调查 蛋白质骨架在这一过程中的作用使用原子规模的突变，并将决定 这些操作通过结构、电生理和计算实验产生的后果。 我们的第二个目标是阐明CLC交换器中的耦合机制。《中图法》运输商 在生物膜上用2Cl-交换1H+。几种致病突变会影响这一过程。 通过未知的机制。我们的目标是阐明CLCs中Cl-/H+偶联的基础。我们将利用 结合常规和原子诱变的计算工具来探测动态 蛋白质进行重排，以形成物理上不同的H+途径 从氯离子所走的路线。第三个目的是确定功能性的分子起源 《中图法》频道与转运体的分歧。尽管有高分辨率的结构 关于这两种亚型的信息，这种功能差异的分子来源仍不清楚。我们将使用 统计系统发育学和进化生物信息学以确定最可能的进化序列 导致功能分歧的事件。然后我们将从功能上刻画重述的序列 这些关键的进化步骤并使用这些信息来识别必要的氨基酸替换子集 以制定功能开关。最终，这些努力将导致新的分子和概念框架 对《中图法》功能的理解，将使我们能够设计出改进的方法 由这些蛋白质的功能失调引起的疾病状态。

ABSTRACT The CLC channels and transporters mediate anion transport through biological membranes. Genes encoding for CLC proteins are found in nearly all organisms, from bacteria to plants and humans. Mutations altering the properties of five of the nine human genes encoding for CLC homologues result in genetic disorders of bone, kidney, brain and muscle, highlighting the fundamental role of these proteins in a wide variety of tissues and cellular compartments. Despite their pathophysiological importance, our understanding of how these proteins function lagged far behind many other classes of ion channels and exchangers. This limits our ability to interpret their function in human physiology and to design targeted pharmacological interventions that would selectively manipulate their activity. Thus, our long-term goal is to elucidate the atomic basis for CLC Cl- channel and transporter function. Our proposal is articulated in three specific aims, each of which addresses a fundamental unanswered mechanistic question on CLC function. Our innovative use of synergistic experimental and computational approaches enables the formulation of specific hypotheses and their rigorous testing. In the first Aim we will determine the bases of substrate selectivity in the CLC Cl- channels. While selectivity of cation channels is well understood, nearly nothing is known on anion selectivity. We will probe the role of the protein backbone in this process using atomic-scale mutagenesis and will determine the consequences of these manipulations through structural, electrophysiological and computational experiments. Our second aim is to elucidate the coupling mechanism in the CLC exchangers. The CLC transporters exchange 2 Cl- for 1 H+ across biological membranes. Several disease-causing mutations affect this process through unknown mechanisms. Our goal is to elucidate the basis for Cl-/H+ coupling in the CLCs. We will utilize computational tools in conjunction to conventional and atomic mutagenesis to probe the dynamic rearrangements undergone by the protein to enable the formation of a pathway for H+ that is physically distinct from the route taken by the Cl- ions. The third aim is to determine the molecular origin of the functional divergence of the CLC channels from the transporters. Despite the availability of high resolution structural information for both subtypes, the molecular origin of this functional divergence remains unknown. We will use statistical phylogenetics and evolutionary bioinformatics to identify the most likely evolutionary sequence of events leading to the functional divergence. We will then functionally characterize sequences recapitulating these key evolutionary steps and use this information to identify a subset of amino acid substitutions necessary to enact the functional switch. Ultimately, these efforts will lead to new molecular and conceptual framework for the understanding of CLC function, which will enable the design of approaches for the amelioration of the disease conditions caused by the dysfunction of these proteins.