Inhibition of the proton-activated chloride channel PAC by PIP2

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    Combining electrophysiology, site-directed mutagenesis, lipid pharmacology, and single particle cryo-electron microscopy, this study provides solid evidence identifying a site on the extracellular half of the transmembrane domain of Proton-Activated Chloride (PAC) channels that could be occupied by PIP2 and related lipids to promote channel desensitization. These findings are important because pharmacological information for these biologically relevant ion channels is absent.

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Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by PACC1 ( TMEM206 ). PAC regulates endosomal acidification and macropinosome shrinkage by releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP 2 at pH 4.0 and identified its putative binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP 2 -binding. Structural and electrophysiological analyses suggest that PIP 2 inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP 2 as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.

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  1. eLife assessment

    Combining electrophysiology, site-directed mutagenesis, lipid pharmacology, and single particle cryo-electron microscopy, this study provides solid evidence identifying a site on the extracellular half of the transmembrane domain of Proton-Activated Chloride (PAC) channels that could be occupied by PIP2 and related lipids to promote channel desensitization. These findings are important because pharmacological information for these biologically relevant ion channels is absent.

  2. Reviewer #1 (Public Review):

    Proton Activated Chloride (PAC) channels have been recently identified as important contributors to endosomal acidification, and their activity in the plasma membrane increases under certain pathological conditions and can induce cellular death. There is very limited information on the pharmacology of these ion channels. By recording from endogenous PAC channels stimulated with an acidic extracellular solution in HEK293 cells using the patch-clamp technique, this study finds that PAC channels are inhibited physiological concentrations of the soluble short-chain PIP2 analog dic-8-PIP2. Inhibition is quantified for several PIP2-related lipids with different number of headgroup phosphates and shorter or longer acyl chains, and it is found that an acyl chain with more than 8 carbons and a negative headgroup charge are both required for robust inhibition. Importantly, inhibition appears to result from PIP2 incorporated into the outer membrane leaflet, as treatment of the inner leaflet with PIP2 or poly-lysine to either increase or decrease PIP2, respectively, did not have any effect on channel activity, as opposed to when the lipid is extracellularly applied. A structure of the channel in the presence of PIP2 was obtained using single-particle cryo-electron microscopy - the structure resembles a previously observed conformation for PAC channels that likely represents a non-conducting desensitized state, and it contains densities with a shape that is consistent with a bound PIP2 molecule in the outer leaflet. Mutations to alanine based on the channel-lipid interactions observed in the structure were all found to disrupt inhibition of PAC channels by PIP2, consistent with the location of the lipid binding site proposed in the study. By comparing the amino acid sequence of human PAC channels with those of other species, it is found that the proposed lipid binding site is highly conserved except in zebrafish. Notably, zebrafish PAC channels are less susceptible to inhibition by PIP2, and mutation of residues at the binding site to those present in the human channel increases inhibition, consistent with the proposed location of the binding site for PIP2. Finally, it is found that the kinetics of inhibition by PIP2 are positively correlated with the degree of channel activation and also with the kinetics of desensitization, suggesting that PIP2 binds more favorably to the desensitized state of the channel whereas it does not bind to the closed state, providing a possible mechanism for the inhibition.

    Results are clearly reported and findings are generally robust. One concern is that most of the electrophysiological characterization of the inhibition of PAC channels by PIP2 lipids was done using endogenously expressed channels. It is unclear why this was done because mutant channels are studied in a PAC KO cell line that could have been used for all experiments. The effects of acidic pH and acidic pH + PIP2 in cells that do not express PAC channels is therefore not shown, but would be important to establish that the measured effects of the lipid are specific to PAC channels.

    Another concern for the study is related to the uncertainty in establishing that the bound lipid is indeed PIP2. Although the mutagenesis results are all consistent with the proposed binding site, it remains a possibility that the mutations affect PIP2 inhibition indirectly by e.g. changing the rate of channel desensitization, which was not measured for any of the mutants on Figure 3E. There is not additional analysis performed to determine whether other types of lipids could occupy the density that is proposed to represent PIP2. Although this might be difficult because no density for the headgroup of the lipid was observed.

    A final caveat of the study is that effects of PIP2 on the extracellular leaflet might be non-physiological. If this were the case, however, the identification of a non-physiological binding site that favors desensitization might still be beneficial for drug design in the context of this channel.

  3. Reviewer #2 (Public Review):

    Proton-activated chloride channel (PAC or ASOR) is a newly discovered anion channel which has a broad tissue expression and is implicated in important physiological processes, such as regulation of endosomal acidification and macropinocytosis. PAC is also implicated in pathological conditions related to acidosis on the plasma membrane. Since its discovery and initial characterization, several structures were solved in resting, activated and desensitized states, revealing an overall channel architecture and its mechanism of action. However, little is known about modulation of PAC channel by endogenous molecules. In the present manuscript, the authors sought to explore the modulation of PAC by lipids, particularly by PIP2, as this lipid is known to modulate numerous unrelated membrane proteins.

    The major strength of the manuscript is the variety of approaches which the authors implement to characterize the mechanism of modulation of PAC by PIP2. Firstly, the authors demonstrate that PIP2 inhibits PAC channel if applied extracellularly. Furthermore, the authors demonstrate that PIP2 acts on the activated/poised towards desensitization, and not on the resting state of the channel. To explore the effect further, the authors tested various PIP molecules, varying in the number of phosphates in the inositol headgroup, and the length of acyl chains. The inhibition of PAC was more potent with the increase of the number of phosphates, and with the lengthening of acyl chains. The lipid chain without inositol, or the inositol without acyl chains, were not as potent in inhibiting PAC. The authors conclude that inositol headgroup together with acyl chains of at least 8 carbons in length are both required to potently inhibit PAC.

    To investigate the potential PIP2 binding site, the authors proceeded to solve the structure of PAC in complex with PIP2. Surprisingly, a density representing a putative PIP2 molecule is found on the extracellular side of the protein. This is a rather unusual finding, given that PIP2 is mostly localized to the inner leaflet of the plasma membrane. To further confirm the binding of PIP2 molecule to this site, the authors mutate the residues interacting with PIP2 molecule in their structure, and observe the decrease in inhibition of the channel by PIP2. Furthermore, the authors observe that these residues are not conserved in all PAC homologs. D. rerio PAC channel does not have these residues and is not inhibited by PIP2 as potently as the human homolog (hPAC). Introducing equivalent residues in D. rerio PAC channel endowed it with modulation by PIP2, similar to hPAC, further strengthening the conclusion that the identified site indeed binds PIP2.

    Overall, the authors succeeded in identifying and characterizing an endogenous molecule with the potential to modulate PAC channel. The present study is the first case of identifying a modulator, characterizing its binding site and mechanism of action on PAC channel. This opens new exciting avenues for structure-guided drug design for this newly-discovered ion channel. However, the localization of the PIP2 binding site to the outer membrane leaflet is quite unexpected, and it is unclear if PAC could be modulated by PIP2 in a physiological context and whether this would be mediated by another lipid transporter. The work will be of interest to ion channel field and a broader membrane protein community with the emphasis on lipid modulation of membrane proteins.

  4. Reviewer #3 (Public Review):

    This compelling manuscript by Mihaljević et al. describes an unusual regulatory mechanism for the proton-activated channel (PAC) where phosphatidylinositol (4,5)-biphosphate (PI(4,5)P2) inhibits the channel by direct interaction with a binding pocket in its extracellular/lumenal domain. This conclusion is supported by electrophysiology data collected on endogenously expressed channels in a human cell line. The authors support their finding with a structural model of acyl groups determined by cryo-electron microscopy. The core experimental design is sound and the data support the narrow conclusions of the paper.

    This manuscript must consider the biological context of PI(4,5)P2 and the relevance of this interaction. Previous studies have documented that PI(4,5)P2 exists on the outer leaflet of the plasma membrane, but as a minor component relative to the overall levels of membrane PI(4,5)P2. The same applies for endosomes, where PIPs are enriched on the cytosolic membrane. The inositol headgroup is unresolved in the structural model of PI(4,5)P2-bound PAC, indicating that this interaction is nonspecific for PI(4,5)P2. This brings up the question as to whether PI(4,5)P2 is the relevant endogenous antagonist for PAC or whether it is a proxy for another ligand that has yet to be determined.