Structural insights into the organization and channel properties of human Pannexin isoforms 1 and 3

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Pannexins are single-membrane large-pore ion channels that release ATP upon activation. Three isoforms of pannexins, 1, 2, and 3, perform diverse cellular roles, including inflammation, differentiation, neuropathic pain, and ATP release. In this study, we report the cryoEM structure of pannexin 3 at 3.9 Å and characterize the structural differences with pannexin isoforms 1 and 2. We observe the organization of the Pannexin 3 vestibule into two distinct chambers with a wider pore radius in comparison to both PANX1 and 2 isoforms. We further report the structure of pannexin1 congenital mutant R217H in the resolution range of 3.9 Å. The congenital mutant R217H in transmembrane helix3 (TM3), R217H induce structural changes that leads to a partially closed pore and altered ATP interaction propensities. The channel conductance of the congenital mutant displays weakened voltage sensitivity. The results showcase a complete comparison of the three pannexin isoform structures that along with the structure of Pannexin 1 congenital mutant highlight distinct structural features of pannexin isoforms and the allosteric role of distant substitutions in dictating channel behavior in Pannexin 1.

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  1. Consolidated peer review report (19 October 2022)


    Pannexin hemichannels are a family of important large pore channels involved in ATP release during apoptosis and many other interesting biological processes. In this preprint, the authors present the first structure of pannexin-3 (PANX3) using cryo-EM, which has a similar overall fold to earlier structures of pannexin-1 (PANX1) but with several unique and interesting features within the pore, including regions where the dimensions and electrostatic potentials are quite unique and a more open lateral portal that may also serve as a permeation pathway. The authors also solve the structures of several mutants of pannexin1, including the disease-causing R217H mutation and several mutations of interesting pore-lining residues. The structures are complemented by electrophysiological measurements of ion conduction as well as ATP binding to explore alterations in ion conduction and how permeant anionic molecules like ATP may interact with the pore. The structure of PANX3 is a very important contribution to the field that will be of interest to many scientists, including those working on pannexin channels and other large pore channels. Although functional data are provided to help guide several key interpretations, they are difficult to evaluate as currently presented, and do not directly address key functional differences that may exist between PANX1 and PANX3, such as single channel conductance or ion selectivity.


    Revisions essential for endorsement:

    1. A systematic concern of the functional data is that it is not possible from the data presented to know how much of the current measured corresponds to leak or endogenous currents without showing data for untransfected cells obtained at the same time as PANX channel recordings. This is important to control for variability in the quality of recordings and expression of endogenous channels over different passages of the HEK cells. If representative, the currents from mock transfected cells in Supplementary Fig. 1 are reassuring, but it is important that such measurements are provided in all figures. It would also be important to spell out what mock transfection means. Were these untransfected cells or cells treated with transfection reagents + empty plasmids? This is a particular concern given that the one inhibitor, CBX, may have lower affinity for PANX3 than PANX1 and therefore cannot be used to identify the currents specifically related to PANX expression. It would be much more convincing if the authors could show I-V data throughout for both untransfected and transfected cells, before and after application of CBX, so that readers can get a sense of the current associated with PANX channels and differences in CBX sensitivity. It would also be good to show data at different CBX concentrations if the authors wish to draw conclusions about differences in CBX sensitivity. Michalski et al., 2018 provide one example of how to present functional data for PANX channels that gives the reader the necessary information to understand key findings. The authors should show a dotted line at zero current level when presenting currents like those shown in Supplementary Fig S1. It would also be best to present representative currents along with I-V and G-V data in the main figures. Finally, from the data presented in Supplementary Fig S1 it appears that the quality of voltage-clamp is at times suboptimal as a slow component is evident in the capacitive transients. Do the authors have sufficient data to select a subset of recordings where capacitive transients are single exponential and rapid, indicative of good voltage clamp speed, and to exclusively use those for presenting example recordings and constructing both I-V and G-V relations?

    2. The data for PANX3 channel activity needs to be re-evaluated in light of contrasting data in the literature. Bruzzone et al., 2003 reported that PANX3-expressing frog oocytes do not respond to voltage steps and Michalski et al. 2018 reported that PANX3 is not functional when stimulated with depolarizing membrane voltage steps, even when surface expression was confirmed using surface biotinylation. This is an important point that the authors should try to address as they are the first to provide functional recordings of PANX3. This finding would be more conclusive if the authors could provide data for untransfected cells obtained side-by-side with that for PANX3-expressing cells. Again, providing data to compare key functional properties, such as ion selectivity, would be a valuable contribution to the field.

    3. The lower current density observed for PANX1 R217H needs to be presented as outlined in point 1, but even then, this finding does not necessarily reflect a change in channel conductance as concluded. It could also be due to changes in surface expression, even though the overall protein expression is comparable to wt, as noted. It would be good to control for possible changes in surface expression, better document similar CBX sensitivity, and ideally measure the single channel conductance using single channel recordings. The shift of G-V observed for R217H in Fig 3e is interesting but the change in voltage-sensitivity is not convincing because the mutant G-V does not appear to be approaching saturation like wt. If G-V relations were obtained by simply dividing current by the difference between the test voltage and Vrev, as indicated, it would also be interesting to look at G-V relations by specifically using the CBX-sensitive current component to calculate G. Although CBX sensitivity may indeed vary for different constructs, the CBX-sensitive current is the most likely component to be associated with the PANX channel, and it would be interesting to see how it compares with the overall G-V.

    4. The ATP binding data suggesting a decreased affinity for R217H is interesting, as is the decrease observed for the R128A mutant. The authors describe the 3-fold decrease in affinity for R24A as minimal, when it is similar to the difference noted earlier between PANX1 and PANX3, which is stated to be weaker. Importantly, no functional data are provided to assess whether R24A or R128 are functional, which is critical for understanding changes in ATP affinity. The text on the bottom of page 8 is also confusing because the authors state “We also observe reduced ATP binding, decreased current density and voltage-sensitivity in the mutant channel (Extended Data Fig 5b,c,e)” when it’s not clear what mutant is being referred to. Presumably R217H, but this needs to be clear, and data for R24A and R128A are needed.

    5. The resolution of the present structures (3.75 to 4.29 Å) is quite modest; sufficient to determine the backbone fold but unlikely to contain adequate density for many side chains. The written presentation could be more nuanced and refer to the quality of side chain density in regions where differences between structures are discussed.

    6. In the discussion, the authors propose that PANX3 is open, but by HOLE standards, this would apply to all solved structures of PANX channels. In addition, this conclusion overlooks the fact that the channel has minimal activity at 0 mV, as well as previous proposals about unresolved intracellular regions potentially occluding the pore and preventing permeation. Admittedly the C-terminus where caspase cleavage activates the channel is shorter in PANX3 compared to PANX1, but we still encourage the authors to be more circumspect when speculating about the functional state that their structures represent.

    7. Interpretation of the PANX1 double mutant structure is difficult because PANX2 is only distantly related to PANX1, so it is unlikely that simply swapping two residues could elucidate the structural relationships, including how differences in a specific region of the pore may influence ion permeation or ion selectivity. Perhaps the author could provide a more compelling rationale by acknowledging the limitations and then put forward the mutant as an initial attempt to perturb an interesting region of PANX1 that is different to PANX3. If not, the authors might consider removing this data from the manuscript because it adds little to the study.

    8. More information is required to interpret the ATP-binding assays. What is the relationship between the apparent ATP-dissociation constant and its permeability? Since those assays potentially also include non-specific interactions, additional support for the observed changes in ATP binding affinity could be provided by a negative control measuring binding to a membrane protein which is known not to interact with ATP. Also, the authors might consider using this assay to measure CBX binding for PANX1 and PANX3. A correlation with lower CBX sensitivity for currents would help to support that interpretation.

    9. The preprint contains a number of statements that are speculative because they are not supported by data, which the authors should either remove or tone down. Examples include:

    "Also, the movements of the aromatic group of F58 through its torsion angle can further alter the diameter of this constriction point and facilitate selective access to the pore".

    "PANX isoforms do not display heterogenous oligomeric associations"

    "A comparison with the Alphafold2 model of PANX3 with the experimental model in this study indicates that the N-terminus faces the cytosol. It constricts the channel from the cytoplasmic side, unlike PANX1WT, where the N-terminus lines the pore and interacts partially with the adjacent protomer"

    "S70 and Q76 between two protomers form a hydrogen bond resulting in inter protomeric interactions leading to a stable heptamer"

    "Extracellular loops and EH1 have an average shift of 1.5-2 Å away from the pore, which along with the slight TM1 movement, is most likely responsible for inducing the change in the W74 position by moving the tryptophan indole ring towards the pore, thereby shortening the pore radius by 4 Å"

    “In comparison to the PANX1WT, the W74 shifts by a χ2 torsion angle of nearly 88.3° towards the pore leading to substantial closure of the pore diameter in PANX1 channels and affecting channel properties"

    "The negatively charged surface in PANX3 may facilitate its role as an ER calcium channel"

    Additional suggestions for the authors to consider:

      1. Given the unique electrostatic properties observed in the structure of PANX3 compared to PANX1, it would be interesting to undertake experiments looking at the relative permeabilities of the two channels to different anions and cations. Figure 3 in Michalski et al., 2020 contains measurements that could be useful to compare PANX1, PANX3 and some of the mutants studied here.
    1. In Figure 3.d. and 4.c. the authors describe the channel physiology of PANX1 mutants and compare them with wt. Adding data for wt PANX1 would make it easier for the reader to understand what the authors wish to convey. Further, in Figure 2g, 3d, and 4c, the authors used normalized electrophysiological data to represent channel physiology, which is justified due to the minimal current through such large pore channels. However, because the methods section did not include the point of normalization for those measurements, including this information would provide a better basis for evaluation of those data points. Further as the authors mention in section “PANX3 displays a double-sieve pore organization”, the pore of PANX3 is lined with residues I74 and R75. While residue I74 is easily recognizable, R75 is not depicted. It would be interesting to see the orientation of these residues towards each other to get a better understanding of their placement and how they interact without consulting the deposited map. In addition, in the section “PANX3 displays a double-sieve pore organization”, the authors nicely describe the observation of a separation between TM2 and CTH1. Since the authors suggest the passage of ions through the separated parts, those findings could be further supported by an electrostatics surface representation of this area.

    2. As described in section “PANX3 displays a double-sieve pore organization”, the authors suggest that the additional constriction site observed in PANX3 leads to the separation of two vestibules with significantly different electrostatic properties. The division of the channel pore allows the regulation of both ATP and Ca2+ ion release, particularly as the first vestibule compartment is thought to be important for Ca2+ binding. Therefore, mutations of the surface of the primary vestibule and the second constriction site, followed with electrophysiological characterization of Ca2+ and ATP release by mutants, would allow the authors to further strengthen their hypothesis.

    3. Fig 2 does not do a very good job of illustrating many of the unique features of the external pore of PANX3 that are discussed at the bottom of pg 5 or in the middle of pg6. We suggest the authors work to improve the presentation in the figure to illustrate the unique features they wish to communicate. It would also be good to show a figure somewhere illustrating the unknown densities discussed on pg 6. What is the experimental evidence that the uncharacterized densities are not noise?

    4. Ref 17 should be Michalski et al., 2020

    5. In Fig 5 it would help to use grey or another unique colour for the HOLE representations to more clearly distinguish the pore from the protein.


    Reviewed by:

    Toshi Kawate, Associate Professor, Cornell University, USA: structure and mechanisms of pannexin channels

    Elena Farah Lehmann, graduate student, University of Zurich, Switzerland: structural biology, cryo-EM, large pore channel families

    Kenton J. Swartz, Senior Investigator, NINDS, NIH, USA: ion channel structure and mechanisms, chemical biology and biophysics, electrophysiology and fluorescence spectroscopy

    Curated by:

    Kenton J. Swartz, Senior Investigator, NINDS, NIH, USA

    (This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)