The Gloeobacter violaceus ligand-gated ion channel (GLIC) has been extensively studied by X-ray crystallography and other biophysical techniques. This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel (pLGIC) signal transduction. However, the GLIC is activated by lowering the pH and the location of its putative proton activation site(s) still remain(s) unknown. To this end, every Asp, Glu, and His residue was mutated individually or in combination and investigated by electrophysiology. In addition to the mutational analysis, key mutations were structurally resolved to address whether particular residues contribute to proton sensing, or alternatively to GLIC-gating, independently of the side chain protonation. The data show that multiple residues located below the orthosteric site, notably E26, D32, E35, and D122 in the lower part of the extracellular domain (ECD), along with E222, H235, E243, and H277 in the transmembrane domain (TMD), alter GLIC activation. D122 and H235 were found to also alter GLIC expression. E35 is identified as a key proton-sensing residue, whereby neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically to the orthosteric site, at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD.

该Gloeobacter菜X射线晶体学和其他生物物理技术已广泛研究了配体门控离子通道（GLIC）。这为五元配体门控离子通道（pLGIC）信号转导的一般门控机制提供了重要见识。然而，通过降低pH来活化GLIC，并且其推定的质子活化位点的位置仍然未知。为此，将每个Asp，Glu和His残基单独或组合突变，并通过电生理学进行研究。除突变分析外，还对关键突变进行了结构解析，以解决特定残基是否对质子感应或对GLIC门控的贡献，而与侧链质子化无关。数据显示，位于正构位点以下的多个残基，特别是E26，D32，E35，细胞外结构域（ECD）下部的D122和E122，跨膜结构域（TMD）的E222，H235，E243和H277会改变GLIC激活。发现D122和H235也改变GLIC表达。E35被鉴定为关键的质子感应残基，由此中和其侧链羧酸盐可稳定活性状态。因此，质子活化在多个基因座的水平上变构发生在正构位点，而ECD和TMD之间的偶联界面起关键作用。