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Structures of human pannexin 1 reveal ion pathways and mechanism of gating

Nature volume 584, pages 646–651 (2020)Cite this article

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Abstract

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.

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Fig. 1: Overall architecture of PANX1.
Fig. 2: CBX binding site of PANX1.
Fig. 3: A single PANX1 subunit.
Fig. 4: Channel assembly of PANX1.
Fig. 5: Ion-conducting pathways and channel gating.

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Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-21588 (PANX1(WT)), EMD-21589 (PANX1(ΔCTT)), EMD-21590 (CBX–PANX(ΔCTT))00, EMD-21591 (PANX1(ΔNTH/ΔCTT)), EMD-21592 (CBX–PANX1(ΔNTH/ΔCTT)), EMD-21593 (PANX1(N255A)Hemi), EMD-21594 (PANX1(N255A)Gap), EMD-21595 (apo PANX1), EMD-21596 (Ca2+–PANX1), EMD-21597 (K+–PANX1) and EMD-21598 (SMA–PANX1). Structure models have been deposited in the RCSB Protein Data Bank under accession codes 6WBF (PANX1(WT)), 6WBG (PANX1(ΔCTT)), 6WBI (CBX–PANX(ΔCTT)), 6WBK (PANX1(ΔNTH/ΔCTT)), 6WBL (CBX–PANX1(ΔNTH/ΔCTT)), 6WBM (PANX1(N255A)Hemi) and 6WBN (PANX1(N255A)Gap).

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Acknowledgements

We thank B. Roth for the initial construct screening; G. Zhao and X. Meng for the support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite; the HPC team of VARI for computational support; and D. Nadziejka for technical editing. W.L. is supported by the National Institutes of Health (NIH) (grant R56HL144929). J.D. is supported by a McKnight Scholar Award, a Klingenstein-Simon Scholar Award, a Sloan Research Fellowship in neuroscience and the NIH (grant R01NS111031). Z.R. is supported by an American Heart Association postdoctoral fellowship (grant 20POST35120556).

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Authors and Affiliations

  1. Van Andel Institute, Grand Rapids, MI, USA

    Zheng Ruan, Ian J. Orozco, Juan Du & Wei Lü

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  1. Zheng Ruan
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  2. Ian J. Orozco
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Contributions

W.L. and J.D. initiated and supervised the project. Z.R. performed mutagenesis, purified PANX1, prepared and screened cryo-EM samples and performed cryo-EM data collection and processing and computational simulation. I.J.O. performed electrophysiological experiments. All authors contributed in manuscript preparation.

Corresponding authors

Correspondence to Juan Du or Wei Lü.

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The authors declare no competing interests.

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Peer review information Nature thanks Stephen Brohawn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Purification and biochemical analysis of PANX1.

a, SEC profile of PANX1 purification using GDN. b, SDS–PAGE of purified PANX1–GFP. For gel source data, see Supplementary Fig. 1a. c, FSEC experiment for caspase 3/7 cleavage of PANX1–GFP. GFP absorbance (480 nm) is shown on the y-axis. d, Caspase 7 cleavage of purified PANX1–GFP. Cleavage of CTT results in a peak shift. Tryptophan absorbance (280 nm) is shown on the y-axis. e, SMA solubilization screening of PANX1–GFP using FSEC. Three SMA polymers (SMA25010, SMA30010 and SMA40001) were tested. GFP absorbance (480 nm) is shown on the y-axis. f, SEC profile of PANX1 purification using SMA30010. Tryptophan absorbance (280 nm) is shown on the y-axis. g, Deglycosylation test of PANX1–GFP and PANX1(N255A)–GFP using PNGase F. Bands corresponding to the glycosylated and non-glycosylated PANX1 are indicated. See Supplementary Fig. 1b for gel source data. h, FSEC analysis of the PANX1 mutations used for electrophysiology recordings. Cells expressing PANX1(WT) or mutants were solubilized using GDN. Gain-of-function mutations with lower expression level are labelled. The R75E mutant contains a peak position shifted to the right. i, j, FSEC analysis on extracellular-gate mutations of PANX1 solubilized using GDN (i) or SMA30010 (j). Peak positions of correctly assembled PANX1 and incorrectly assembled PANX1 are indicated by arrows and vertical bars. The W74R, R75D and R75E mutants showed decreased stability relative to wild type because, when extracted using detergent, they mostly ran at positions representing incorrect assemblies (i). Nevertheless, the SMA-extracted W74R, R75D and R75E mutants still showed peaks at positions representing correct assemblies (j), indicating that they are able to form correctly assembled channel complex in a native lipid environment.

Extended Data Fig. 2 The workflow of cryo-EM data processing of PANX1 constructs that do not form gap junctions.

a, The data-analysis pipeline for PANX1 cryo-EM analysis with no gap junction. Special attention was paid to determine whether the dataset indeed adheres to C7 symmetry. Two examples of PANX1 adopting a symmetric conformation (PANX1(ΔCTT)) or a non-symmetric conformation (CBX–PANX1(ΔCTT)) are shown. A detailed description of the data-analysis procedure can be found in Methods. b, The overlay of the C1 refined maps of PANX1(ΔCTT), CBX–PANX1(ΔCTT), PANX1(ΔNTH/ΔCTT) and CBX–PANX1(ΔNTH/ΔCTT) with the symmetric model of PANX1. The CBX–PANX1(ΔCTT), PANX1((ΔNTH/ΔCTT)) and CBX–PANX1(ΔNTH/ΔCTT) maps adopt a non-symmetric shape.

Extended Data Fig. 3 Representative micrographs, 2D class averages and Fourier shell correlation curves for all datasets in this study.

a–j, For each dataset, a representative micrograph, four 2D class averages and Fourier shell correlation (FSC) curve plot are shown, except for PANX1(N255A) dataset, in which two structures are shown. The map resolution is determined on the basis of the gold-standard 0.143 criterion. If an atomic model is available for the dataset, a model versus map FSC curve is also provided. The model versus map resolution is determined on the basis of the 0.5 FSC criterion. In a, a slice view of the SMA–PANX1 map showing the organization of the TMD is shown.

Extended Data Fig. 4 Local resolution estimation and representative densities.

a, b, PANX1 map. c, d, The PANX1(N255A)Gap map. To provide better visualization on the exterior and interior map quality, both non-sliced and sliced views of the maps are shown. The colour represents the local resolution in Å. Representative densities are shown for a few selected secondary structure elements of PANX1.

Extended Data Fig. 5 The workflow of cryo-EM data processing for PANX1(N255A).

To separate gap junction particles from hemichannel particles, we relied on 2D classification to distinguish tilted and side views. The top and down views were separated during 3D classification. A more detailed description of the data-analysis pipeline can be found in the Methods.

Extended Data Fig. 6 The structures of PANX1(ΔNTH/ΔCTT).

a, The apo state. b, PANX1(ΔNTH/ΔCTT) in complex with CBX. CBX is shown in orange. In a, b, odd- and even-numbered subunits are shown in blue and white, respectively; the seventh subunit is in green. Cryo-EM maps viewed parallel to the membrane (left) and from the extracellular side (middle). The unsharpened map is shown as a transparent envelope. Right, structural models viewed from the intracellular side. c, The slice view of the extracellular entrance of PANX1(ΔNTH/ΔCTT) (left) and CBX–PANX1(ΔNTH/ΔCTT) (right) maps. The CBX molecule and the side chain of W74 are shown in stick.

Extended Data Fig. 7 Patch-clamp analysis of the wild-type PANX1 and its mutants.

a, Representative traces of whole-cell current density from patch-clamped tsA201 control cells (n = 6) and tsA201 cells overexpressing: wild type (n = 12) and ΔNTH (n = 5), W74A (n = 8), R75E (n = 5), N255A (n = 5), Δ21–23 (n = 6), Δ21–27 (n = 7), R29A (n = 9), A33W (n = 10), A33W/ΔCTT (n = 4), ΔCTT (n = 4) and A33C (n = 5) mutant PANX1. Voltage steps (0.25 s) of 20 mV were imposed from −100 mV to +80 mV from a holding potential of −10 mV. Cells were first measured in standard bath solution and then re-measured following the superfusion of a bath solution containing 0.1 mM carbenoxolone. b, Mean current measured at 5 ms of experiments in a plotted as a function of clamp voltage. c, Current amplitudes of experiments in a with and without CBX. Each paired point represents an individual cell and the bar represents the mean. d, Plot of zero-current reversal potentials using a 2-s voltage ramp for various bath solutions containing either (in mM): 145 NaCl, 145 NaI, 145 sodium gluconate, 14.5 NaCl or 145 NMDG-Cl (see Methods for complete solutions). The number of cells patched (indicated in parenthesis) for the various bath solutions were as follows, 145 mM NaCl: wild type (5), R29A (7), W74A (5), R75E (7); 145 mM NaI: wild type (5), R29A (7), W74A (5), R75E (7); 145 mM sodium gluconate: wild type (5), R29A (7), W74A (5), R75E (7); 14.5 mM NaCl: wild type (4), R29A (4), W74A (5), R75E (5); 145 mM NMDG-Cl: wild type (3), R29A (2), W74A (5), R75E (3). e, f, Plots of the calculated (Methods) permeability of iodide relative to chloride (PI/PCl), the permeability of gluconate relative to chloride (Pgluconate/PCl) and permeability of sodium relative to chloride (PNa/PCl) for wild-type and mutant channels. For statistical comparisons to wild type, one-way analysis of variance with Bonferroni correction was performed. For PI/PCl, P = 0.99 (R29A), 3.7 × 10−5 (W74A) and 5.9 × 10−5 (R75E). For Pgluconate/PCl, P = 0.046 (R29A), 0.99 (W74A) and 6.8 × 10−9 (R75E). For PNa/PCl, P = 0.99 (R29A), 0.99 (W74A) and 2.2 × 10−6 (R75E). Each point represents an individual cell. The bar represents mean and error bars show s.e.m.

Extended Data Fig. 8 Comparison of large-pore channels.

a, The structures of large-pore channels, viewed parallel (top) or perpendicular (bottom) to the membrane. One subunit (or one pair of subunits) is in green. The diameter of VRAC is calculated without the cytoplasmic leucine-rich repeat domain. b, Organization of the TMD, viewed from the intracellular side. The NTH and transmembrane helices S1, S2, S3 and S4 are labelled for two subunits. Only CALHM2 has its transmembrane helices arranged in a clockwise manner. The contact between adjacent TMDs in PANX1 is made by the NTH with the S1 and S2 helices in the neighbouring subunit; the same contact in CALHM2 is made by the S2 and S4 of adjacent subunits, and in connexin by the S1 and S2 of adjacent subunits. There is no major contact in innexin and VRAC.

Extended Data Fig. 9 Secondary structure arrangement and sequence alignment.

Secondary structures based on the PANX1 structure model are labelled. The W74 forming the extracellular entrance is marked with an arrow. Key residues forming the side tunnel are labelled with a red asterisk. The cysteine residues forming the extracellular disulfide bonds are highlighted by an orange dot. The N255 glycosylation site is marked with a green dot. The gap junction interface and caspase 3/7 cleavage site are indicated with a red frame. A gain-of-function disease mutation (Δ21–23) of PANX1 is also marked.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics for PANX1(WT), apo PANX1(WT), Ca2+–PANX1(WT) and K+–PANX1(WT)
Full size table
Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics for PANX1(ΔCTT), CBX–PANX1(ΔCTT), PANX1(ΔNTH/ΔCTT) and CBX–PANX1(ΔNTH/ΔCTT)
Full size table
Extended Data Table 3 Cryo-EM data collection, refinement and validation statistics for PANX1(N255A)Hemi, PANX1(N255A)Gap, SMA–PANX1(WT)
Full size table

Supplementary information

Supplementary Figure 1

This file contains the raw gel images for Extended Data Fig. 1b and 1g.

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Ruan, Z., Orozco, I.J., Du, J. et al. Structures of human pannexin 1 reveal ion pathways and mechanism of gating. Nature 584, 646–651 (2020). https://doi.org/10.1038/s41586-020-2357-y

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