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

ABSTRACT: 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.

SUBMITTER: Yang J

PROVIDER: S-EPMC7305803 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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

Yang Junhua J Chen Jianan J Del Carmen Vitery Maria M Osei-Owusu James J Chu Jiachen J Yu Haiyang H Sun Shuying S Qiu Zhaozhu Z

Science (New York, N.Y.) 20190425 6438

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 gen ...[more]

PMID: 31023925

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

Project description: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.

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Proton-activated chloride channel PAC regulates endosomal acidification and transferrin receptor-mediated endocytosis.

Project description:During vesicular acidification, chloride (Cl-), as the counterion, provides the electrical shunt for proton pumping by the vacuolar H+ ATPase. Intracellular CLC transporters mediate Cl- influx to the endolysosomes through their 2Cl-/H+ exchange activity. However, whole-endolysosomal patch-clamp recording also revealed a mysterious conductance releasing Cl- from the lumen. It remains unknown whether CLCs or other Cl- channels are responsible for this activity. Here, we show that the newly identified proton-activated Cl- (PAC) channel traffics from the plasma membrane to endosomes via the classical YxxL motif. PAC deletion abolishes the endosomal Cl- conductance, raises luminal Cl- level, lowers luminal pH, and increases transferrin receptor-mediated endocytosis. PAC overexpression generates a large endosomal Cl- current with properties similar to those of endogenous conductance, hypo-acidifies endosomal pH, and reduces transferrin uptake. We propose that the endosomal Cl- PAC channel functions as a low pH sensor and prevents hyper-acidification by releasing Cl- from the lumen.

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Molecular Insights into the Inhibition of Proton-Activated Chloride Channel by Transfer RNA

Project description:The proton-activated chloride (PAC) channel (also known as acid-sensitive outwardly rectifying anion channel, ASOR) plays a critical role in acid-induced cell death and endocytosis. However, little is known about the regulatory factors and binding partners of PAC. In this study, we discovered that transfer RNA (tRNA) interacts directly with PAC as an unexpected binding partner. Using cryo-electron microscopy, we determined that two PAC trimers and one co-purified tRNA molecule form a stable complex via a highly conserved KR motif, representing the closed conformation. tRNA is located on the intracellular side of PAC, blocking the channel pores. Furthermore, electrophysiological data showed that tRNA modulates chloride currents and channel open probability of PAC, thus protecting against acid-induced cell death. Our study provides insight into the regulation of PAC activity by cytosolic tRNA and extends the role of tRNAs in pathological and physiological events.

2024-06-28 | GSE216637 | GEO

Cryo-EM structure of a proton-activated chloride channel TMEM206.

Project description:TMEM206 has been recently identified as an evolutionarily conserved chloride channel that underlies ubiquitously expressed, proton-activated, outwardly rectifying anion currents. Here, we report the cryo-electron microscopy structure of pufferfish TMEM206, which forms a trimeric channel, with each subunit comprising two transmembrane segments and a large extracellular domain. An ample vestibule in the extracellular region is accessible laterally from the three side portals. The central pore contains multiple constrictions. A conserved lysine residue near the cytoplasmic end of the inner helix forms the presumed chloride ion selectivity filter. Unprecedentedly, the core structure and assembly closely resemble those of the epithelial sodium channel/degenerin family of sodium channels that are unrelated in amino acid sequence and conduct cations instead of anions. Together with electrophysiology, this work provides insights into ion conduction and gating for a new class of chloride channels that is architecturally distinct from previously characterized chloride channel families.

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

Project description: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 injury1-3. PAC has recently been shown to represent a novel and evolutionarily conserved protein family4,5. 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.

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A new polymodal gating model of the proton-activated chloride channel.

Project description:The proton-activated chloride (PAC) channel plays critical roles in ischemic neuron death, but its activation mechanisms remain elusive. Here, we investigated the gating of PAC channels using its novel bifunctional modulator C77304. C77304 acted as a weak activator of the PAC channel, causing moderate activation by acting on its proton gating. However, at higher concentrations, C77304 acted as a weak inhibitor, suppressing channel activity. This dual function was achieved by interacting with 2 modulatory sites of the channel, each with different affinities and dependencies on the channel's state. Moreover, we discovered a protonation-independent voltage activation of the PAC channel that appears to operate through an ion-flux gating mechanism. Through scanning-mutagenesis and molecular dynamics simulation, we confirmed that E181, E257, and E261 in the human PAC channel serve as primary proton sensors, as their alanine mutations eliminated the channel's proton gating while sparing the voltage-dependent gating. This proton-sensing mechanism was conserved among orthologous PAC channels from different species. Collectively, our data unveils the polymodal gating and proton-sensing mechanisms in the PAC channel that may inspire potential drug development.

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Ion permeation controlled by hydrophobic residues and proton binding in the proton-activated chloride channel.

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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Gating choreography and mechanism of the human proton-activated chloride channel ASOR.

Project description:The proton-activated chloride channel ASOR (TMEM206/PAC) permeates anions across cellular membranes in response to acidification, thereby enhancing acid-induced cell death and regulating endocytosis. The molecular mechanisms of pH-dependent control are not understood, in part because structural information for an activated conformation of ASOR is lacking. Here, we reconstitute function from purified protein and present a 3.1-Å-resolution cryo-electron microscopy structure of human ASOR at acidic pH in an activated conformation. The work contextualizes a previous acidic pH structure as a desensitized conformation. Combined with electrophysiological studies and high-resolution structures of resting and desensitized states, the work reveals mechanisms of proton sensing and ion pore gating. Clusters of extracellular acidic residues function as pH sensors and coalesce when protonated. Ensuing conformational changes induce metamorphosis of transmembrane helices to fashion an ion conduction pathway unique to the activated conformation. The studies identify a new paradigm of channel gating in this ubiquitous ion channel.

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Project description:To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.

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