Molecular mechanism underlying desensitization of the human proton-activated chloride (PAC) channel.

Abstract

Opened by acidic pH, the Proton-Activated Chloride (PAC) channel has been recorded in a variety of cell types. Although its molecular identify has only recently been identified, the PAC channel has established important roles in acid-induced cell death, endosomal acidification, and macropinosome shrinkage. Previous cryo-EM studies of the PAC channel reported a proton-bound non-conducting structure at low pH, suggestive that PAC undergoes desensitization - a process common to ligand-gated ion channels. Comparison of the proposed open and desensitized structures demonstrated significant rotational movement of the transmembrane helix 1 (TM1), switching its interaction partner to a neighboring subunit. However, properties and underlying mechanisms underlying PAC channel desensitization remain to be largely unknown. In this study, we demonstrate that PAC undergoes pH-dependent desensitization upon prolonged acid exposure with patch-clamp electrophysiology. Additional studies with structure-guided mutagenesis 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 TM1, as well as E107, D109, and E250 at the interface of the extracellular domain (ECD) and transmembrane domain (TMD). 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. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for further investigation of its relevance in cellular physiology and disease.