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Abstract：
CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a \"ball-and-chain\" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.
摘要：
CLC-2是一种电压门控氯离子通道，有助于许多不同哺乳动物组织和细胞类型的电兴奋性和离子稳态。在九种哺乳动物CLC同源物中，CLC-2被超极化唯一激活,而不是去极化，质膜。密切相关的CLC同源物之间电压门控机制极性差异的分子基础一直是一个长期的谜。部分原因是可用的CLC通道结构很少，和那些存在的表现出高度的构象相似性。这里,我们报告了人类CLC-2在2.46-2.76µ的冷冻EM结构，在存在和不存在有效和选择性抑制剂AK-42的情况下。AK-42在Cl-渗透途径的细胞外入口通道内结合，占据了以前通过计算对接研究提出的口袋。在apo结构中，我们观察到CLC-2的两种不同的apo构象，涉及细胞质C末端结构域（CTDs）之一的旋转。在没有CTD轮换的情况下，细胞内N末端15个残基的发夹肽与TM结构域嵌套，以物理方式从细胞内侧阻塞Cl-渗透途径。该肽在CLC-2的物种变体中高度保守,但不存在于任何其他CLC同源物中。先前的研究表明，CLC-2的N端域通过“球链”门控机制影响通道特性，但是相互矛盾的数据使人们对这种机制产生了怀疑，因此，N端结构域的结构及其与通道的相互作用一直不确定。通过对缺乏15个残基发夹肽的N端缺失突变体的电生理研究，我们表明，这种短序列的丢失会增加哺乳动物细胞中表达的CLC-2电流的大小并减少其整流。此外，我们表明，在重复超极化的情况下，WTCLC-2电流与发夹缺失的CLC-2电流相似。这些功能结果与我们的结构数据相结合，支持了一个模型，其中CLC-2的N末端发夹通过阻断细胞质Cl-渗透途径来稳定通道的闭合状态。
