Different cell types, such as HEK-293T and MDCK, transiently transfected with the Myc-tagged N254Q mutant showed a predominant intracellular localization (Number 1)

Different cell types, such as HEK-293T and MDCK, transiently transfected with the Myc-tagged N254Q mutant showed a predominant intracellular localization (Number 1). connexins. The temporal variations of Panx1 trafficking correlate with spatial variations of intracellular localizations induced by Golgi-blockage by Brefeldin-A or glycosylation prevention by tunicamycin. Consequently, Panx1 offers kinetics and dynamics that make it unique to serve unique functions independent from connexin-based channels. oocytes pairs indicated that while Panx1 can form junctional channels (Boassa et al., 2007; Bruzzone et al., 2003), it does so at much reduced levels than connexins in either oocytes (Boassa et al., 2007) or in N2A cells (Penuela et al., 2007) implying the functional form of Panx1 is definitely a hemichannel (pannexon) in one plasma membrane. It has recently been reported that unlike connexins, murine Panx1 is definitely unaffected by Brefeldin A treatment (Penuela et al., 2007) and converts over more slowly than connexins (Boassa et al., 2007; Penuela et al., 2007). In addition, varying amounts of cell surface expression of the N254Q mutant were also reported (Boassa et al., 2007; Penuela et al., 2007). Specifically, in our studies we used Myc-tagged N254Q mutant rPanx1 while Penuela and collaborators analyzed untagged mPanx1 suggesting the differences observed could R406 besylate be due to the effect of the tag on the protein. Following up on our observations, we investigated the trafficking dynamics of native, mutant and tagged versions R406 besylate of Panx1. We display that cell surface expression levels of the N254Q mutant are rescued by co-expression with rPanx1 wild-type (WT) proteins. This rescue, however, was limited to the amount of unglycosylated Panx1 in the plasma membrane and did not result in an increase in space junction formation. On the other hand, we display that de-glycosylation with the glycosidase PNGase F resulted in significantly higher junctional conductances in pairs of oocytes expressing Panx1. Furthermore, using different pharmacological providers we analyzed the trafficking kinetics of tagged rPanx1 versus WT following inhibition of protein glycosylation or membrane traffic and secretion. It is well known that connexins have a short half-life estimated at 1.5-5 hours depending on the cell type (Laird, 2006) and that tagged connexins traffic at a slightly slower rate than WT (Jordan et al., 1999). While native Panx1 becomes over significantly slower than connexins, here we demonstrate that in contrast to tagged connexins, Myc or tetracysteine (referred to as 4C) tagged Panx1 move more quickly through the cells. Methods Plasmids and mutagenesis The cDNAs encoding rat Panx1 wild-type, -myc tagged were kindly provided by Dr. Roberto Bruzzone. Site-directed mutagenesis was done with the R406 besylate Quikchange kit (Stratagene, La Jolla, CA) as previously explained (Boassa et al., 2007). Cell tradition and transfections Human being embryonic kidney (HEK) -293T and Madin-Darby canine kidney (MDCK) cells were managed at Gimap6 37C, and 10 %10 % CO2 in Dulbeccos Modified Eagles medium comprising 10% fetal bovine serum (GIBCO-BRL, Invitrogen, Carlsbad, CA). Transfections were performed by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transductions were carried out using a retroviral system according to the protocols from your Nolan laboratory (www.stanford.edu/group/nolan). Experiments were carried out either 1-2 days after transient transfection or on stably expressing cell lines generated by transduction followed by selection with the antibiotic hygromycin (Gibco-BRL, Invitrogen). Antibodies We used the following main antibodies: chicken anti-Panx1 (4515, characterized in (Locovei et al., 2006); mouse monoclonal anti-myc (Sigma, St. Louis, MO); rabbit polyclonal anti-giantin (Covance Study Products, Denver, PA). Immunocytochemistry and confocal microscopy Cells were cultivated on poly-D-lysine-coated glass coverslips, fixed in 4% paraformaldehyde/phosphate buffered saline for 20 moments, washed and labeled for immunofluorescence. The primary antibodies (as explained in number legends) were combined and diluted with obstructing buffer diluted 5-fold in PBS. The secondary antibodies (fluorescein isothiocyanate-conjugated anti-mouse or anti-chicken, Cy5-conjugated anti-mouse and rhodamine red-X anti-rabbit) were diluted 1:100 in the same buffer. Data acquisition was done with an Olympus FluoView1000 laser-scanning confocal microscope (Olympus, Center Valley, PA). Electrophysiology The oocyte cell-cell channel assay was performed as explained earlier (Dahl, 1992). The follicle coating was removed from oocytes by collagenase treatment. The oocytes were injected with transcribed Panx1 cRNA and incubated for 2-3 days. The vitelline membrane was eliminated with forceps and the oocytes combined. Junctional conductance was identified with the dual voltage clamp technique (Aerosol et al., 1981) 6 hours after pairing. Pharmacological treatment of oocytes consisted of 30 minute incubations treated with either soybean glycine maximum (10 g/ml, Sigma, St. Louis, MO), tunicamycin (1 g/ml, Sigma, St. Louis, MO), or PNGase F (10 devices/ml, New.