人源氯离子通道ClC-2和CFTR的结构生物学研究

细胞膜对于维持细胞正常生理功能是必不可少的，细胞膜上的转运蛋白和离子通道蛋白对于吸收营养物质，排出代谢废物等有害物质，以及维持细胞内外离子和水的平衡非常重要。氯离子是生物体内含量最丰富的阴离子，不仅中和钠、钾、钙等阳离子的正电荷，维持细胞整体电中性，还参与调节细胞的渗透压、表皮组织的粘液分泌、维持肌肉组织和神经组织的可兴奋性。细胞内外氯离子浓度的动态平衡，是氯离子通道蛋白和氯离子转运蛋白等共同调控的结果。
人体主要的氯离子通道包括囊性纤维化相关膜蛋白ABCC7(CFTR)、电压门控氯离子通道ClC蛋白家族、GABA和glycine配体门控氯离子通道等。研究表明，这些氯离子通道的功能异常会引起多种遗传疾病。本课题选取了CFTR氯离子通道蛋白和ClC蛋白家族的几个成员作为研究对象，通过生物化学和结构生物学等手段，研究调节剂的作用机理，为研发靶向这些人源氯离子通道的药物提供分子层面的理论基础。经过多次尝试和优化冷冻电镜样品制备条件，本课题最终获得中等分辨率CFTR氯离子通道结构和高分辨率ClC-2氯离子通道结构。
ClC-2是在人体各组织器官里广泛表达的一种电压门控的氯离子通道。其功能减退的遗传突变会引起视网膜退化、雄性不育、大脑白质病变等症状；其功能增强型突变能够导致醛固酮增多等内分泌紊乱症状，与高血压有关联。目前已知有两个特异性的ClC-2抑制剂：来源于蝎子毒素的GaTx2小肽和人工合成的小分子化合物AK-42；但还没有报道过特异性ClC-2激活剂。
本研究表达纯化了全长人源ClC-2 蛋白，经过多次优化冷冻电镜制样条件和数据处理过程，获得了ClC-2氯离子通道同源二聚体在apo状态与结合高效抑制剂AK-42状态的冷冻电镜结构，整体分辨率均为约4.3 Å，跨膜区分辨率为约3.5 Å。首先对apo状态ClC-2蛋白的氯离子运输路径的半径、门控机制以及氯离子结合位点等性质进行了分析：该ClC-2结构里，门控谷氨酸侧链占据离子通道的中间氯离子结合位点，快门控处于关闭状态，与离子选择性有关的保守丝氨酸和酪氨酸在离子通道半径狭窄处发挥作用。我们进一步探究了AK-42抑制ClC-2活性的机理：AK-42结合在通道的外侧氯离子结合位点上方，直接堵塞了氯离子跨膜运输路径。随后，分析了ClC-2和抑制剂的相互作用：AK-42可以和ClC-2蛋白的三个氨基酸形成氢键，和一个氨基酸有阳离子-π相互作用，此外还和多个氨基酸有疏水相互作用。此外，本课题通过分子动力学模拟进行能量分解研究，定量分析了每个氨基酸对结合AK-42的能量贡献。参与结合AK-42的氨基酸位点，在其他人源ClC家族蛋白对应的氨基酸序列上至少有5个差异，这也解释了AK-42能够特异性抑制ClC-2，而不影响其他人源ClC蛋白活性。最后，通过构建结合位点突变体，并使用膜片钳进行电生理实验，分析了突变对AK-42抑制ClC-2效果的影响，细胞水平的电生理实验支持了结构生物学的分析结论。综上所述，本研究为理解人源ClC-2氯离子通道的门控特点，以及与AK-42抑制剂作用机理奠定了基础，有助于研发靶向人源ClC-2氯离子通道的药物。
此外，还表达纯化了CFTR氯离子通道蛋白，计划获得它与抑制剂CFTRinh-172的复合物结构，已获得约6 Å分辨率密度图，两个NBD结构分开，12次跨膜螺旋形成的两个TMD结构呈V字形向内开口，通道呈关闭状态，两个TMD之间有4处密度差异，揭示了潜在的抑制剂结合位点。
综上所述，虽然ClC-2和CFTR这两个氯离子通道在门控机制方面有所不同，但都会受到ATP浓度影响，而且它们在氯离子选择性滤器上有较高相似性；同时ClC-2蛋白的特异性抑制剂AK-42和CFTR蛋白特异性抑制剂CFTRinh-172都通过直接堵塞氯离子运输路径来发挥作用。这两个蛋白的结构生物学研究，有助于深化我们对于氯离子通道的理解，对于依赖结构的药物设计与优化具有启发意义。

The cell membranes are essential for cells to maintain normal physiological functions. The transporters and channels on the membrane are crucial to absorb nutrients, export cellular toxic metabolic wastes and keep the balance of ions and water. As the most plentiful anion in organisms, chloride is counterion for sodium, potassium and calcium cations, it not only keeps the cellular electroneutrality, but also ensures the osmotic balance of cells, transepithelial transport and excitability of muscle and nervous tissues. The thermodynamic equilibrium of cellular chloride concentration is affected by factors such as chloride channels and transporters.
Human chloride channels mainly include cystic fibrosis related membrane protein ABCC7(CFTR), voltage regulated chloride channels ClC and (GABA or glycine) ligand gated channels. The malfunctions of chloride channels can cause many genetic disorders. In order to solve the structures of chloride channels in complex with their modulators, and investigate the mechanisms of channel modulators for drug discovery, the CFTR chloride channel and several ClC members have been selected as study subjects. After many trials and optimizations of cryo-EM grids preparation, we have ultimately gotten medium-resolution CFTR cryo-EM density and high-resolution ClC-2 structures. 
The human ClC-2 is a widely expressed voltage gated chloride channel. Its loss-of-function mutations can lead to retina degeneration, azoospermia and leukodystrophy. In contrast, elevated aldosterone and higher blood pressure are associated with ClC-2 gain-of-function variations. There are no ClC-2 specific activators till now, while it has two reported specific inhibitors: GaTx2 peptide from scorpion venom and synthetic small compound AK-42.
In this study, we have expressed and purified full-length human ClC-2 protein. After the optimization of the cryo-EM grids preparations and data processing, we have solved high resolution structures of ClC-2 homodimer in apo and AK-42 inhibitor binding states, the overall resolution is 4.3 Å, while the resolution of transmembrane domain is 3.5 Å. Firstly, the radius, gating mechanism and chloride binding site of ClC-2 channel have been analyzed: the gating glutamate has occupied the central chloride binding site in our ClC-2 structures, the selectivity filter formed by conserved serine and tyrosine residue has located in the narrowest place of chloride channel. Next, the mechanism of AK-42 inhibition on ClC-2 have been analyzed: the AK-42 inhibitor has located above the extra chloride binding site and directly blocked the chloride channel. Then, we have studied the interactions between ClC-2 and AK-42: the inhibitor can form hydrogen bonds with 3 amino acids of proteins, cation-π interaction with one amino acid, and hydrophobic interactions with several amino acids. Then the molecular dynamics simulation has been used to do energy decomposition study of AK-42 binding amino acids. These amino acids have at least 5 differences in other human ClC family proteins, which also explains the specific inhibition of AK-42 on ClC-2, with no obvious influence on other human ClC protein. Finally, we have utilized the patch clamp technique to do cellular level validations for interactions between AK-42 and (wildtype or mutant) ClC-2, the electrophysiological experiments have supported our structural biology conclusions. In summary, this work will increase the understanding of human ClC-2 chloride channel gating and inhibition by AK-42, which may be helpful on the development of ClC-2 targeting drugs.
In addition, in order to solve the structure of CFTR and CFTRinh-172, I have purified the CFTR protein, and gotten 6 Å resolution map. The two NBDs were separated, the two TMDs formed by the 12 transmembrane helices opened inward in a V-shape, and the channel was closed. There were 4 suspected inhibitor binding sites between the two TMDs.
 In summary, although the two chloride channels ClC-2 and CFTR are different in gating mechanisms, they are regulated by ATP, and they have high similarities in selective filters. Both ClC-2 inhibitor AK-42 and CFTR inhibitor CFTRinh-172 act by directly blocking the chloride transport pathway. The structural study of these two proteins will increase our understanding of chloride channels and provide insights for structure-based drug design and optimization.
 

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