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Structures and pH-sensing mechanism of the proton-activated chloride channel.

ABSTRACT: The proton-activated chloride channel (PAC) is active across a wide range of mammalian cells and is involved in acid-induced cell death and tissue injury^1-3. PAC has recently been shown to represent a novel and evolutionarily conserved protein family^4,5. Here we present two cryo-electron microscopy structures of human PAC in a high-pH resting closed state and a low-pH proton-bound non-conducting state. PAC is a trimer in which each subunit consists of a transmembrane domain (TMD), which is formed of two helices (TM1 and TM2), and an extracellular domain (ECD). Upon a decrease of pH from 8 to 4, we observed marked conformational changes in the ECD-TMD interface and the TMD. The rearrangement of the ECD-TMD interface is characterized by the movement of the histidine 98 residue, which is, after acidification, decoupled from the resting position and inserted into an acidic pocket that is about 5 Å away. Within the TMD, TM1 undergoes a rotational movement, switching its interaction partner from its cognate TM2 to the adjacent TM2. The anion selectivity of PAC is determined by the positively charged lysine 319 residue on TM2, and replacing lysine 319 with a glutamate residue converts PAC to a cation-selective channel. Our data provide a glimpse of the molecular assembly of PAC, and a basis for understanding the mechanism of proton-dependent activation.

SUBMITTER: Ruan Z

PROVIDER: S-EPMC7773282 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recently identified proton-activated chloride channel (PAC) contains two transmembrane helices (S1-S2) and is involved in lysosome function, hypoxia adaption, stroke, and carcinogenesis. Although a PAC structure was recently resolved, its gating and activation mechanisms remained largely unknown. By the two-electrode voltage clamp electrophysiology in Xenopus oocytes, we found that the hydrophobicity of site 304 at fenestrations, but not that of neighbor sites, is important for maintaining PAC at a closed state at pH 7.5. When activated at acidic pH, PAC activity significantly increased with the hydrophilicity of site 307 within S2, but not with that of neighbor sites, suggesting that 307 acts as an activation gate. We identified six conserved protonatable residues critical for proton-induced activation, consistent with structural studies. Our study depicted a scheme in which proton binding induces conformational changes from the W304-controlled closed state at fenestrations to an activated state controlled by activation gate I307 in helix S2.

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PAC, an evolutionarily conserved membrane protein, is a proton-activated chloride channel.

Project description:Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl- (PAC) currents (I Cl,H). Overexpression of human PAC in PAC knockout cells generated I Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl- channel family in physiological and pathological processes associated with acidic pH.

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