Inhibition of the proton-activated chloride channel PAC by PIP2
- Johns Hopkins Medicine, United States
- Van Andel Institute, United States
Abstract
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 (PIP2) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP2 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 PIP2-binding. Structural and electrophysiological analyses suggest that PIP2 inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP2 as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.
Data availability
The cryo-EM density map and atomic models have been deposited in the EMDB (Electron Microscopy Data Bank) and the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCS-PDB), respectively, under accession numbers EMD-28535 and PDB 8EQ4.
-
Cryo-EM structure of PAC channel with PIP2Electron Microscopy Data Bank, EMD-28535.
-
Cryo-EM structure of PAC channel with PIP2Electron Microscopy Data Bank, EMD-28964.
-
Cryo-EM structure of PAC channel with PIP2Protein Data Bank, 8EQ4.
-
Cryo-EM structure of PAC channel with PIP2Protein Data Bank,8FBL.
Article and author information
Author details
Funding
Boehringer Ingelheim Fonds (Graduate Student Fellowship)
- Ljubica Mihaljevic
National Institutes of Health (grant R35GM124824)
- Zhaozhu Qiu
National Institutes of Health (grant R01NS118014)
- Zhaozhu Qiu
National Institute of General Medical Sciences (T32 GM007445 Graduate Training Program)
- Ljubica Mihaljevic
American Heart Association (Postdoctoral Fellowship grant 20POST35120556)
- Zheng Ruan
National Institutes of Health (grant K99NS128258)
- Zheng Ruan
American Heart Association (Predoctoral Fellowship grant 18PRE34060025)
- James Osei-Owusu
National Institutes of Health (grant R01NS112363)
- Wei Lü
McKnight Foundation (McKnight Scholar Award)
- Zhaozhu Qiu
Alfred P. Sloan Foundation (Sloan Research Fellowship)
- Zhaozhu Qiu
Esther A. and Joseph Klingenstein Fund (Klingenstein-Simons Fellowship)
- Zhaozhu Qiu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Mihaljevic et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,288
- views
-
- 318
- downloads
-
- 5
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Inhibition of the proton-activated chloride channel PAC by PIP2
eLife 12:e83935.
https://doi.org/10.7554/eLife.83935
Further reading
-
- Structural Biology and Molecular Biophysics
Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets from mouse retina using cryo-electron tomography. We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two intertwined coiled coils. The rootlet’s filamentous architecture, with frequent membrane-connecting cross-striations, lends itself well for anchoring large membranes in the cell.
-
- Structural Biology and Molecular Biophysics
Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, local spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.
