Molecular mechanism underlying desensitization of the proton-activated chloride channel PAC

Abstract

Desensitization is a common property of membrane receptors, including ion channels. The newly identified proton-activated chloride (PAC) channel plays an important role in regulating the pH and size of organelles in the endocytic pathway, and is also involved in acid-induced cell death. However, how the PAC channel desensitizes is largely unknown. Here, we show by patch-clamp electrophysiological studies that PAC (also known as TMEM206/ASOR) undergoes pH-dependent desensitization upon prolonged acid exposure. Through structure-guided and comprehensive mutagenesis, we identified several residues critical for PAC desensitization, including histidine (H) 98, glutamic acid (E) 94, and aspartic acid (D) 91 at the extracellular extension of the transmembrane helix 1 (TM1), as well as E107, D109, and E250 at the extracellular domain (ECD)–transmembrane domain (TMD) interface. Structural analysis and molecular dynamic simulations revealed extensive interactions between residues at the TM1 extension and those at the ECD–TMD interface. These interactions likely facilitate PAC desensitization by stabilizing the desensitized conformation of TM1, which undergoes a characteristic rotational movement from the resting and activated states to the desensitized state. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for future investigation of its relevance in cellular physiology and disease.

Inhibition of the proton-activated chloride channel PAC by PIP2

“…Since PIP 2 -bound PAC structure resembles the desensitized state and the PAC channel exhibits apparent desensitization at pH 4.0 ( Osei-Owusu et al, 2022b ) we sought to examine how pH may influence PIP 2 -mediated PAC inhibition by applying diC 8 -PIP 2 at pH 4.0 when the currents stabilized after the initial fast desensitization ( Figure 4A and B ). The percentage of PAC inhibition by PIP 2 increased significantly at pH 4.0 compared with pH 5.0 ( Figure 4C ).…”

“…The proton binding to the ECD is directly coupled with the channel opening in the transmembrane domain (TMD) ( Osei-Owusu et al, 2022a ). After prolonged exposure to pH 4.6 or below, the PAC channel slowly desensitizes ( Osei-Owusu et al, 2022b ). The desensitization of PAC is pH-dependentthat is, under more acidic conditions, desensitization is stronger ( Osei-Owusu et al, 2022b ).…”

“…After prolonged exposure to pH 4.6 or below, the PAC channel slowly desensitizes ( Osei-Owusu et al, 2022b ). The desensitization of PAC is pH-dependentthat is, under more acidic conditions, desensitization is stronger ( Osei-Owusu et al, 2022b ). This is regulated by several key residues localized at the ECD–TMD ( Osei-Owusu et al, 2022b ).…”

“…The desensitization of PAC is pH-dependentthat is, under more acidic conditions, desensitization is stronger ( Osei-Owusu et al, 2022b ). This is regulated by several key residues localized at the ECD–TMD ( Osei-Owusu et al, 2022b ). For example, the E94R mutant displays fast desensitization even at pH 5.0, when the wild-type channel does not exhibit obvious current decay ( Osei-Owusu et al, 2022b ).…”

“…This is regulated by several key residues localized at the ECD–TMD ( Osei-Owusu et al, 2022b ). For example, the E94R mutant displays fast desensitization even at pH 5.0, when the wild-type channel does not exhibit obvious current decay ( Osei-Owusu et al, 2022b ). In addition to the resting and desensitized structures ( Ruan et al, 2020 ), an open conformation of PAC was recently reported ( Wang et al, 2022 ).…”