Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel

The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl2channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conserva-tive negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton -sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.

Automated summary

The protein TMEM206 is the molecular basis of the extracellular proton-activated Cl2 channel (PAC), which plays a crucial role in neuronal death during ischemia-reperfusion. The mechanism of proton sensitivity in the PAC channel is unclear, but two acidic amino acid residues were found to form a proton-sensitive site in the channel. Chloral hydrate was also found to activate the channel, and knockout mice resistant to chloral hydrate-induced sedation and hypnosis were observed. This study provides insights into the activation mechanism of the hPAC channel and identifies a potential drug target in chloral hydrate.