Expression and localization of panx1a WT and mutants in Neuro 2a cells. (A) Sequence alignment of panx1a with pannexin orthologs and paralogs and the Caenorhabditis elegans INX-6 protein. (B) Topological representation of a panx1a subunit showing the positions of both aromatic residues at the transmembrane and intracellular loop borders. The image was generated using Protter [27]. (C) Localization of EYFP-tagged wild type panx1a and mutants in transfected Neuro 2a cells. Scale bar: 10µm. (D) Western blot analysis of transfected EYFP-tagged panx1a and mutants. The three bands correspond to nonglycosylated panx1a (Gly0), the high mannose glycosylation (Gly1), and the complex glycosylation (Gly2) forms. Expressed proteins were detected using an anti-GFP antibody and an anti-ß-actin antibody as the control.

Comparison of the panx1a Y205F and Y205A mutants. (A) Neuro 2a cells transfected with WT panx1a-EYFP (left) and the Y205F-EYFP mutant (right) both showed membrane localization. Scale bar: 10µm. (B) Western blot analysis revealed similar glycosylation patterns when the Y205F mutant was compared to WT panx1a. (C) Cell surface biotinylation assay showing that Y205F was present at the membrane of transfected Neuro 2a cells. Cell lysates after NP40 buffer lysis (Input) showed expression of both intracellular (ß-actin; Y205A) and membrane proteins (panx1a; Y205F). The streptavidin pull-down fractions (Biotinylation) showed that all intracellular proteins were depleted, with panx1a and Y205F found in the protein fraction from the cell surface. ß-actin served as an internal control, showing no bands in the biotinylation fraction. Expressed proteins were detected using an anti-GFP antibody and an anti-ß-actin antibody as the control.

Functional analysis of the Y205F mutant using dye uptake assay and Fluorescence Recovery After Photobleaching (FRAP). (A) Fluorescent images are showing dye uptake of panx1a WT and mutants 10 min after EtBr application. EYFP-transfected cells were used as a negative control. Scale bar: 10µm. (B) Total EtBr fluorescence measured 10 min post dye application shown as a bar graph. Wild type panx1a: n = 17, Y205A: n = 14, Y205F: n = 16, W123A: n = 13, EYFP: n = 13. (C) EtBr fluorescence measured throughout the 10 min with one-minute intervals. (D) FRAP analysis of WT panx1a and Y205F mutant showing selected regions (white rectangles) pre bleaching, immediately after bleaching, and recovery 5 s and 50 s post bleaching. Scale bar: 10um. (E) Total % recovery, 50 s post photobleaching. Wild type panx1a: n = 35, Y205F: n = 33. (F). % recovery measured throughout 50 s with five-second intervals. Error bars show the standard deviation of the mean. ***p < 0.001, N.S., not significant.

Expression of Brefeldin A (BFA)-treated cells, co-localization with cellular markers, and ER stress analysis of panx1a and the Y205A mutant. (A) Images of panx1a-EYFP transfected Neuro 2a cells treated with 5ug/mL BFA for 19h showed a reduction of cell membrane localization. Scale bar: 10µm. (B) Western blot analysis showed that treatment with 5ug/mL BFA for 19h caused a decrease in the Gly2 state and an increase in the Gly1 state of the wild type panx1a protein. No effect was detected for the Y205A mutant. Proteins were detected using an anti-GFP antibody. ß-actin served as a protein loading control. (C, D) Co-localization of EYFP-tagged panx1a and the Y205A mutant with mCherry-tagged Sec24D (COP II vesicle marker), His-tagged Cav-1 (Caveolin-1 marker), DsRed-tagged calreticulin (ER marker), and DsRed-tagged galactosyltransferases (Golgi apparatus marker). His-tagged Cav-1 was detected using an anti-His antibody and Alexa Fluor 568 secondary antibody. (E, F) Co-localization quantification of the wild type and mutant panx1a with the vesicle and organelle markers. Error bars show standard error of the mean. WT panx1a + Sec24D: n = 34, WT panx1a + Cav-1: n = 36, WT panx1a + ER: n = 29, WT panx1a + Golgi: n = 35, Y205A + Sec24D: n = 30, Y205A + Cav-1: n = 31, Y205A + ER: n = 20, Y205A + Golgi: n = 20. (G) Real-time qPCR analysis of ER stress genes. Neuro 2a cells were transfected with EYFP alone, panx1a-EYFP, and Y205A-EYFP. When indicated, cells were treated with 5ug/mL BFA for 19h. 18s rRNA was used as the reference gene. EYFP-transfected cells were used as the control group. Data were collected in three independent experiments in triplicate for each gene. All data were relative to the EYFP control group. ****p < 0.0001, ***p < 0.001, *p < 0.05.

Interaction studies of panx1a and the Y205A mutant using FRET and pull-down assay. (A) Fluorescent image showing a Neuro 2a cell transfected with panx1a-DsRed and Y205A-EYFP. Scale bar: 10µm. (B) FRET efficiencies. WT–WT (membrane) (n = 103) indicates panx1a-DsRed interaction with panx1a-EYFP using selected cell membrane regions as a positive control. WT–WT (intracellular) (n = 100) indicates panx1a-DsRed interaction with panx1a-EYFP using selected regions inside the cell. WT-Y205A (n = 63) indicates panx1a-DsRed interaction with Y205A-EYFP inside the cell. Y205A-Y205A (n = 50) indicates Y205A-DsRed interaction with Y205A-EYFP inside the cell. DsRed-EYFP indicates DsRed interaction with EYFP inside the cell as the negative control. The dotted line represents the FRET efficiency threshold of 1.4%. (C) His60 Ni pull-down using cells double-transfected with panx1a-dTomato-His and panx1a-EYFP, Y205A-dTomato-His and panx1a-EYFP, or dTomato-His with panx1a-EYFP. Cell lysates on the left show expression levels before pull down. Elutions on the right show proteins recovered by pull down. Anti-GFP and anti-His antibodies detected eluted proteins. An anti-ß-actin antibody served as a loading control. ****p < 0.0001, N.S., not significant.