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. 2020 Feb 12;9:e54670. doi: 10.7554/eLife.54670

Figure 1. frPanx1 forms a heptameric ion channel.

(a) Whole-cell patch clamp recordings from HEK 293 cells expressing hPanx1, frPanx1, and frPanx1-ΔLC. Cells were clamped at −60 mV and stepped from −100 mV to +100 mV for 1 s in 20 mV increments. To facilitate electrophysiological studies, we inserted a Gly-Ser motif immediately after the start Met to enhance Panx1 channel opening as we previously described (Michalski et al., 2018). CBX (100 μM) was applied through a rapid solution exchanger. (b) Current-voltage plot of the same channels shown in (a). Recordings performed in normal external buffer are shown as circles, and those performed during CBX (100 μM) application are shown as squares. Each point represents the mean of at least three different recordings, and error bars represent the SEM. (c) EM map of frPanx1-ΔLC shown from within the plane of the membrane. Each protomer is colored differently, with the extracellular side designated as ‘out’ and the intracellular side as ‘in.’ (d) Overall structure of frPanx1-ΔLC viewed from within the lipid bilayer. (e) Structure of frPanx1 viewed from the extracellular face.

Figure 1.

Figure 1—figure supplement 1. Sequence alignment and structural features.

Figure 1—figure supplement 1.

Amino acid sequence of frPanx1 compared to various Panx1 orthologues. Amino acids highlighted in black are completely conserved, in gray are similar, and in white are not conserved. Disulfide-forming cysteines are highlighted in yellow. Secondary structure features are shown above the sequence alignment. Green boxes depict the transmembrane helix boundaries, and orange boxes show deleted or truncated regions to create frPanx1-ΔLC.
Figure 1—figure supplement 2. Characterization of frPanx1-ΔLC.

Figure 1—figure supplement 2.

(a) Size exclusion chromatogram of frPanx1-ΔLC. Concentrated protein was injected onto a Superose 6 10/300 column equilibrated with 150 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM DDM. (b) SDS-PAGE analysis of peak fractions collected from (a). (c) Size exclusion chromatogram of frPanx1-ΔLC after reconstitution into nanodiscs. The running buffer contained 150 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA. (d) SDS-PAGE analysis of peak fractions collected from (c). (e) Whole-cell recordings of wild-type (no Gly-Ser) hPanx1, frPanx1, and frPanx1-ΔLC. Whole-cell patches from transfected HEK 293 T cells were obtained, held at −60 mV, and stepped between −100 mV and +160 mV for 1 s. CBX (100 μM) was applied through a rapid solution exchanger. (f) Current-voltage plot of wild-type pannexin recordings shown in (e). Each circle represents the mean current at a particular voltage, with squares depicting the same current when treated with 100 μM CBX. N = 3–11.
Figure 1—figure supplement 3. Cryo-EM image processing workflow for single particle analysis of frPanx1-ΔLC.

Figure 1—figure supplement 3.

(a) A representative micrograph (scale bar = 50 nm), representative 2D class averages, and the 3D classification workflow are shown. (b) The FSC plots of the two half maps (top) and the map vs model (bottom) are shown. (c) The angular distribution plot for class 3. (d) Local resolutions of class three were calculated using ResMap (Kucukelbir et al., 2014).
Figure 1—figure supplement 4. Representative cryo-EM density of frPanx1-ΔLC.

Figure 1—figure supplement 4.

Each domain is shown as stick representation and fit into the corresponding density contoured at σ = 3.0.