ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2. However, experimental structures are still missing to decipher its inhibition mechanism. Here, we present cryo-EM structures of apo ClC-2 and its complex with AK-42, both at 3.5 Å resolution. Residues S162, E205 and Y553 are involved in chloride binding and contribute to the ion selectivity. The side-chain of the gating glutamate E205 occupies the putative central chloride-binding site, indicating that our structure represents a closed state. Structural analysis, molecular dynamics and electrophysiological recordings identify key residues to interact with AK-42. Several AK-42 interacting residues are present in ClC-2 but not in other ClCs, providing a possible explanation for AK-42 specificity. Taken together, our results experimentally reveal the potential inhibition mechanism of ClC-2 inhibitor AK-42.

ClC-2 跨质膜转运氯离子，在细胞稳态中发挥关键作用。其功能障碍与脑白质营养不良和原发性醛固酮增多症等疾病有关。最近报道 AK-42 是 ClC-2 的特异性抑制剂。然而，仍然缺乏实验结构来破译其抑制机制。在这里，我们展示了 apo ClC-2 及其与 AK-42 的复合物的冷冻电镜结构，分辨率均为 3.5 Å。残基 S162、E205 和 Y553 参与氯离子结合并有助于离子选择性。门控谷氨酸 E205 的侧链占据了假定的中心氯离子结合位点，表明我们的结构代表封闭状态。结构分析、分子动力学和电生理记录确定了与 AK-42 相互作用的关键残基。多个 AK-42 相互作用残基存在于 ClC-2 中，但不存在于其他 ClC 中，这为 AK-42 特异性提供了可能的解释。综上所述，我们的结果通过实验揭示了 ClC-2 抑制剂 AK-42 的潜在抑制机制。