Molecular determinants of pH sensing in the proton-activated chloride channel.

The acidic environment is critical for the proper function of many intracellular organelles. It is also associated with various diseases such as ischemia, cancer, and inflammation. In response to acidic pH, the proton-activated chloride (PAC) channel permeates chloride ions across membranes and plays an important role in endosomal acidification. It is also involved in acid-induced cell death and ischemic brain injury. However, how proton binding leads to the opening of this newly identified family of ion channels remains unknown. In this study, we identified several critical protonation sites and intersubunit interactions that work together to determine PAC pH sensitivity. Our work reveals a distinct pH-sensing mechanism and is relevant to the development of inhibitors targeting PAC in diseases associated with acidosis. In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an important role in both cellular physiology (endosome acidification) and diseases associated with tissue acidosis (acid-induced cell death). Despite the available structural information, how proton binding in the extracellular domain (ECD) leads to PAC channel opening remains largely unknown. Here, through comprehensive mutagenesis and electrophysiological studies, we identified several critical titratable residues, including two histidine residues (H130 and H131) and an aspartic acid residue (D269) at the distal end of the ECD, together with the previously characterized H98 at the transmembrane domain–ECD interface, as potential pH sensors for human PAC. Mutations of these residues resulted in significant changes in pH sensitivity. Some combined mutants also exhibited large basal PAC channel activities at neutral pH. By combining molecular dynamics simulations with structural and functional analysis, we further found that the β12 strand at the intersubunit interface and the associated “joint region” connecting the upper and lower ECDs allosterically regulate the proton-dependent PAC activation. Our studies suggest a distinct pH-sensing and gating mechanism of this new family of ion channels sensitive to acidic environment.

酸性环境对许多细胞内细胞器的正常功能至关重要。它还与多种疾病相关，如缺血、癌症和炎症。响应酸性pH，质子激活的氯离子（PAC）通道使氯离子透过细胞膜，在内体酸化过程中发挥重要作用。它还参与酸诱导的细胞死亡和缺血性脑损伤。然而，质子结合如何导致这个新发现的离子通道家族开放仍然未知。在这项研究中，我们确定了几个关键的质子化位点和亚基间相互作用，它们共同决定了PAC的pH敏感性。我们的工作揭示了一种独特的pH感应机制，并且与在酸中毒相关疾病中针对PAC开发抑制剂有关。 响应酸性pH，广泛表达的质子激活的氯离子（PAC）通道开放，使阴离子穿过细胞膜。通过这种方式，PAC在细胞生理学（内体酸化）和与组织酸中毒相关的疾病（酸诱导的细胞死亡）中都发挥着重要作用。尽管有可用的结构信息，但细胞外结构域（ECD）中的质子结合如何导致PAC通道开放在很大程度上仍然未知。在这里，通过全面的诱变和电生理研究，我们确定了几个关键的可滴定残基，包括ECD远端的两个组氨酸残基（H130和H131）和一个天冬氨酸残基（D269），以及先前在跨膜结构域 - ECD界面鉴定的H98，它们是人类PAC潜在的pH传感器。这些残基的突变导致pH敏感性发生显著变化。一些组合突变体在中性pH下也表现出较大的基础PAC通道活性。通过将分子动力学模拟与结构和功能分析相结合，我们进一步发现亚基间界面的β12链以及连接上下ECD的相关“连接区域”变构调节质子依赖性PAC激活。我们的研究表明了这个对酸性环境敏感的新离子通道家族独特的pH感应和门控机制。