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First published online 9 October 2007
doi: 10.1242/jcs.009514

Journal of Cell Science 120, 3772-3783 (2007)
Published by The Company of Biologists 2007
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Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins

Silvia Penuela1, Ruchi Bhalla1, Xiang-Qun Gong2, Kyle N. Cowan3, Steven J. Celetti2, Bryce J. Cowan4, Donglin Bai2, Qing Shao1 and Dale W. Laird1,*

1 Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, N6A 5C1, Canada
2 Department of Physiology and Pharmacology, University of Western Ontario, London, ON, N6A 5C1, Canada
3 Department of Surgery, University of Western Ontario, London, ON, N6A 5C1, Canada
4 Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada


Figure 1
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  		Fig. 1. Sequence analysis and cloning of mouse Panx1 and Panx3. (A) Sequence alignment of mouse Panx1 and Panx3. ClustalW alignment and BLAST analysis of the two protein sequences showed 41% identity at the amino acid level (black boxes). Some regions of high homology coincide with the four transmembrane domains (TM1-TM4) predicted by the Toppred algorithm. Asterisks mark conserved cysteine residues present in both predicted extracellular loops. Underlined sequences in the carboxyl-tail indicate the peptides used for the generation of polyclonal antibodies for each pannexin. Gray boxes indicate conserved amino acid substitutions. (B) Based on sequence analysis, both Panx1 and Panx3 are predicted to be polytopic and contain several predicted N-glycosylation (red and orange residues) and phosphorylation (black) sites. Orange residues indicate extracellular N-glycosylation sites used for site-directed mutagenesis. (C) RT-PCR products generated with specific primers designed to amplify the entire coding regions of each pannexin. A 1.5 kb band corresponding to Panx1 was amplified from both brain and osteoblast RNA from 3-week-old mice. A 1.4 kb Panx3 product was amplified from osteoblast RNA. All bands were sequenced to verify their identity as pannexin transcripts, but only the clones containing the entire coding region were engineered into expression vectors for further analysis.

 

Figure 2
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  		Fig. 2. Panx1 and Panx3 are localized to the cell surface when expressed in NRK cells. Confocal microscopic images of NRK cells expressing untagged Panx1 or Panx3 (A) or GFP-tagged Panx1 or Panx3 (B). Immunolabeling with mouse (anti-Panx1) and human (anti-hPANX1) antibodies revealed that Panx1 and Panx1-GFP were found at the cell surface with increased intensity at cell-cell interfaces when both cells expressed Panx1 or Panx1-GFP. Similar to that observed for Panx1, Panx3 displayed cell surface localization. However, Panx3-GFP was retained within the cell and colocalized with protein disulfide isomerase, an endoplasmic reticulum protein. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Antibody specificity and Panx1 and Panx3 species. Affinity-purified anti-Panx1 antibody shows high specificity to Panx1 both in immunolabeling (A) and western blot (B) when pre-adsorbed with cognate peptides. (B) Multiple species of Panx1 at ~41-48 kD and Panx1-GFP at ~70-80 kD were detected. (C,D) Affinity-purified anti-Panx3 antibody revealed high specificity to Panx3 both in immunolabeling (C) and western blot (D) when pre-absorbed with cognate peptides. Only one species of Panx3 or Panx3-GFP was detected at ~43 kD or ~70 kD, respectively. Anti-GAPDH was used to assess gel loading. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Unlike Cx43-GFP, cell surface Panx1 and Panx3 localization is unaffected by BFA treatment. BICR-M1Rk cells co-expressing Cx43-GFP and Panx1 (A) or Panx3 (B) were treated with 5 µg/ml BFA for 6 hours. After treatment, Cx43-GFP plaques internalized from the cell surface and newly biosynthesized Cx43-GFP accumulated within intracellular compartments, whereas the localization of Panx1 and Panx3 remained unchanged. The experiment shown is representative of three independent repeats. Bars, 20 µm.

 

Figure 5
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  		Fig. 5. Panx1 expression levels were unchanged during cycloheximide treatment. BICR-M1Rk cells expressing endogenous Cx43 and exogenous Panx1 were exposed to 20 µg/ml of CHX for 2, 4, 6 and 8 hours and immunoblotted for Panx1 (A) and Cx43 (B). When compared to un-treated cells (0 hr), immunoblot analysis of Panx1 revealed no significant difference (P=0.4980) between the banding profile at 2, 4, 6 and 8 hours of CHX treatment. However, given the known short half-life of Cx43, the level of Cx43 consistently diminished following 2, 4, 6 and 8 hours of CHX treatment. beta-actin was used as a protein loading control. The experiment shown is representative of three independent repeats. WT, wild type.

 

Figure 6
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  		Fig. 6. Panx1 and Panx3 are highly glycosylated. (A) Whereas the multiple species of Cx43 were reduced to only one species upon alkaline phosphatase treatment, western blotting revealed that the multiple species of Panx1 remained unchanged by the same treatment. (B) Panx1 and Panx3 were found to be glycosylated, as revealed by protein-band shifts upon digestion with 10 units of N-glycosidase F. The glycoprotein transferrin was used as a positive control for N-glycosidase activity and anti-GAPDH antibody was used to assess gel loading.

 

Figure 7
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  		Fig. 7. Localization profile of N-glycosylation-defective mutants of Panx1 and Panx3. (A) A sub-population of N-glycosylation-defective pannexin mutants localized to the cell surface (red) but these proteins also exhibited an increase in intracellular localization when compared with their wild-type counterparts. Notice that specific anti-pannexin labeling was eliminated by pre-incubating the antibodies with cognate peptide. (B,C) Western blots revealed that the Panx1N254Q and Panx3N71Q mutants exist as species that are insensitive to 10 U of N-glycosidase treatment. Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Tissue expression of Panx1 and Panx3. Affinity-purified anti-Panx1 (A) and anti-Panx3 (B,C) antibodies were used to examine the expression of Panx1 and Panx3 in several tissue types from 3-week-old mice and in the thymus from neonatal mice. (A) Panx1 was prevalent in many murine organs and exhibited variable degrees of apparent glycosylation (~41-48 kD), whereas two putative isoforms of Panx3 (~43 kD and ~70 kD) were observed in skin, cartilage and heart ventricle (B). (C) Only a ~70 kD band was detected with the anti-Panx3 antibody in lung, liver, spleen and thymus, with a notable ~50 kD band present in kidney, whereas brain tissue exhibited little or no detectable Panx3. The tissues and Panx3 control used in C were obtained from the same gel. A peptide pre-adsorption assay eliminated most of the staining both for the Panx1 and Panx3 antibodies, indicating that these antibodies are specific for Panx1 and Panx3, respectively. Anti beta-actin or anti-GAPDH was used as a loading control. MW, molecular weight standards; Ventricle, heart ventricle; Panx1, Panx1-expressing NRK cells; Panx3, Panx3-expressing NRK cells.

 

Figure 9
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  		Fig. 9. Localization of endogenous Panx1 and Panx3 in normal human skin and mouse spleen. (A-F) Representative images of immunofluorescent labeling for Cx43, Panx1 and Panx3 (red) in normal human facial skin samples and counterstained with the Hoechst nuclear stain (blue). Labeling with either an antibody raised against mouse (A) or human (C) Panx1 identified Panx1 in a focal pattern distributed throughout the epidermis, but most abundantly in the layers of the stratum granulosum and spinosum. (D)Peptide competition with the human PANX1 antibody revealed the loss of specific labeling. (B) Cx43, localized for comparison, was present focally within the upper layers of the epidermis. (E,F) Immunolabeled Panx3 displayed a diffuse cellular distribution within the epidermis (E) that was eliminated by peptide competition (F). Broken lines denote the extent of the cornified layer; dotted lines indicate the edge of the epidermal vital layer. (G,H) Panx1 was localized in 3-week-old normal mouse spleen (G, red), whereas this staining pattern was eliminated after pre-adsorption with cognate peptide (H). Insert in H shows hematoxylin and eosin staining of a parallel spleen section. Bars, 20 µm.

 

Figure 10
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  		Fig. 10. Panx1 and Panx3 assemble into functional single-membrane channels in 293T cells but do not form functional intercellular channels in N2A cells. (A) Fluorescent micrographs of wild-type (WT) and pannexin-expressing 293T cells after sulforhodamine B dye uptake (left column: 20x objective). The middle column denotes the corresponding phase-contrast images, which includes the background uptake of the large control dye, dextran-rhodamine (inserts). (Right column) In parallel experiments, immunolabeling for pannexins (red) and counterstaining with Hoechst dye (blue) revealed the high pannexin transfection efficiency (63x oil objective). (B) GJIC-deficient N2A cells were engineered to transiently express Cx43-GFP, Cx26-GFP, Panx1-GFP or Panx3-GFP, or to co-express Panx1 or Panx3 and DsRed. Isolated N2A cell pairs with clear green or red fluorescence were chosen for dual whole-cell patch-clamp recordings 48 hours later to measure intercellular junctional conductance. The expression of Cx43-GFP and Cx26-GFP resulted in robust electrical coupling conductance between N2A cells, which was significantly higher than the coupling observed in paired control wild-type N2A (wt N2A) cells. However, both Panx1- and Panx3-expressing N2A cells exhibited no significant increase in electrical coupling above what was observed in wild-type N2A cells. Data were presented as mean±s.e.m.; **P<0.01. The number presented over each column represents the n value for each experimental condition.

 

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© The Company of Biologists Ltd 2007