Pannexin1 Channels Contain a Glycosylation Site That Targets the Hexamer to the Plasma Membrane

Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues. Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues. Intercellular communication takes place in two different ways: release of molecules such as neurotransmitters, hormones, or ATP; and formation of intercellular channels that connect directly the cytoplasm of adjacent cells. Multicellular organisms have evolved to use different types of intercellular channels, called gap junctions. In vertebrates, a gap junction channel is formed by the apposition of two hexameric assemblies of connexins (Cx), 2The abbreviations used are: Cx, connexins; Panx, pannexins; Inx, innexins; DSP, dithio-bis(succinimidylpropionate); PNGase F, N-glycosidase F; Endo H, endoglycosidase H; CIAP, calf intestinal alkaline phosphatase; MDCK, Madin-Darby canine kidney; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein.2The abbreviations used are: Cx, connexins; Panx, pannexins; Inx, innexins; DSP, dithio-bis(succinimidylpropionate); PNGase F, N-glycosidase F; Endo H, endoglycosidase H; CIAP, calf intestinal alkaline phosphatase; MDCK, Madin-Darby canine kidney; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein. one from each adjacent cell. A new class of proteins analogous to connexins in their folding, but completely unrelated in sequence has been identified in invertebrates. These proteins were named innexins (invertebrate connexin analogs, abbreviated as Inx). Despite the conserved structure, properties, and functions of gap junctions in vertebrates and invertebrates, connexin and innexin structures evolved independently. Mammalian analogs of innexins were recently identified in the human genome sequence and the term pannexins (from the Greek word pan, “throughout” and the Latin nexus, “connection,” abbreviated as Panx) was coined to name this new class of proteins (1Panchin Y. Kim I. Matz M. Lukyanov K. Usman N. Lukyanov S. Curr. Biol. 2000; 10: R473-R474Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Pannexins have some sequence similarity to invertebrate innexin proteins, but share no homology with vertebrate connexins. It has been speculated that there is an evolutionary relationship between pannexins and innexins. Both groups of proteins are predicted to have four transmembrane regions, two extracellular loops and intracellular amino (NH2) and carboxyl (COOH) termini. The human pannexin family consists of three members: Pannexin1 (Panx1, 426 amino acids, 47.6 kDa), Pannexin2 (Panx2, 664 amino acids, 73.3 kDa), and Pannexin3 (Panx3, 392 amino acids, 44.7 kDa). Pannexins are expressed in many different systems and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins have been proposed to form gap junction-like structures (1Panchin Y. Kim I. Matz M. Lukyanov K. Usman N. Lukyanov S. Curr. Biol. 2000; 10: R473-R474Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Earlier studies by Bruzzone et al. (2Bruzzone R. Kim S.G. Barbe M.T. Herb A. Monyer H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13644-13649Crossref PubMed Scopus (614) Google Scholar) in paired Xenopus oocytes found that the expression of Panx1, alone and in combination with Panx2, induces intercellular channels. However, functional and localization studies in erythrocytes, cultured neurons, and glia have shown only plasma membrane staining with no evidence of canonical gap junctions (3Locovei S. Kim L. Dahl G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7655-7659Crossref PubMed Scopus (421) Google Scholar, 4Huang Y. Kim J.B. Abrams C.K. Scherer S.S. Glia. 2007; 55: 46-56Crossref PubMed Scopus (148) Google Scholar). One current hypothesis is that Panxs do not duplicate the function of Cxs in connecting directly the cytoplasm of adjacent cells, but rather connect the cytoplasm to the extracellular space. The hemichannels formed by Panx1 in the plasma membrane have been shown to be mechanosensitive ATP conduits (5Bao L. Kim S. Dahl G. FEBS Lett. 2004; 572: 65-68Crossref PubMed Scopus (618) Google Scholar) that respond to several signal transduction pathways. They are involved in the release of small molecules, and may promote the propagation of calcium waves (6Locovei S. Kim J. Dahl G. FEBS Lett. 2006; 580: 239-244Crossref PubMed Scopus (415) Google Scholar, 7Pelegrin P. Kim A. EMBO J. 2006; 25: 5071-5082Crossref PubMed Scopus (1126) Google Scholar, 8Pelegrin P. Kim A. J. Biol. Chem. 2007; 282: 2386-2394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 9Locovei S. Kim E. Qiu F. Spray D.C. Dahl G. FEBS Lett. 2007; 581: 483-488Crossref PubMed Scopus (353) Google Scholar, 10Litvin O. Kim A. Connell-Alberts Y. Panchin Y. Baranova A. J. Cell Mol. Med. 2006; 10: 613-634Crossref PubMed Scopus (53) Google Scholar). In this study, we investigated the stoichiometry and membrane targeting of Panx1 oligomers. Panx1 is widely expressed in several tissues, and the expression pattern varies in different locations. For example, Panx1 revealed postsynaptic localization in rodent hippocampal and cortical principal neurons (11Zoidl G. Kim E. Ray A. Meier C. Bunse S. Habbes H.W. Dahl G. Dermietzel R. Neuroscience. 2007; 146: 9-16Crossref PubMed Scopus (122) Google Scholar). Furthermore, Panx1-specific labeling was evident in the perinuclear region of retinal ganglion cells and horizontal cells, whereas relatively little plasma membrane staining was observed in neonatal retinal ganglion cells, gradually increasing with age (12Dvoriantchikova G. Kim D. Panchin Y. Shestopalov V.I. FEBS Lett. 2006; 580: 2178-2182Crossref PubMed Scopus (84) Google Scholar). To uncover the mechanism regulating the localization of pannexins, in particular subcellular compartments, might provide insights into the functional role of these novel channel proteins in different tissues. The data presented here show for the first time that Panx1 forms a multimeric channel and contains a glycosylation site at its extracellular surface that is important for the trafficking of the protein to the cell surface. The presence of a glycosylation site argues that the single hexamer is the primary channel state in contrast to connexin-based structures that are formed by two docked hexamers where each hexamer can act as an independent unit (13Verselis V.K. Kim E.B. Bukauskas F.F. Braz. J. Med. Biol. Res. 2000; 33: 379-389Crossref PubMed Scopus (41) Google Scholar). Antibodies—We used the following primary antibodies: chicken anti-Panx1 (4515, characterized in Ref. 3Locovei S. Kim L. Dahl G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7655-7659Crossref PubMed Scopus (421) Google Scholar); mouse monoclonal anti-Myc (Sigma); mouse anti-α-tubulin (Sigma); and rabbit polyclonal anti-calnexin (Stressgen, Victoria, Canada). 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 QuikChange kit (Stratagene, La Jolla, CA) using Pfu Turbo DNA polymerase. Complementary pairs of synthetic oligonucleotide primers containing the desired mutation were used to amplify the full-length plasmid with the rPanx1 cDNA insert. The PCR product was treated with DpnI endonuclease to digest the methylated wild-type DNA template, and transformed into MAX Efficiency DH5α Competent Cells (Invitrogen). Successful incorporation of the mutations was confirmed by DNA sequence analyses, using forward and reverse primers to check the entire rPanx1 sequence, and confirm the absence of random mutations. Cell Culture and Transfections—HEK-293T cells were maintained at 37 °C, and 10% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen). Plasmids (0.5 μg for transfection of 35-mm dish) were into cells using (Invitrogen). cells were of Culture cells were in and on a and at for at The was in on and with to a of The was on for the The was at for at in a in a The was and on for the used the to Panx1 the The was use in and to a of or and at The was by of and on for A of of was to the The were analyzed by or and with anti-Myc and were as by et al. H. Kim J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). were from cells in containing and a on a and to a membrane were in and in and for with primary chicken anti-Panx1 mouse anti-α-tubulin or mouse monoclonal anti-Myc were with an kit The of was using the of and from cells Panx1 wild-type or proteins were with of endoglycosidase or of N-glycosidase for at 37 at and or with of calf intestinal alkaline for at 37 and were on in for and for The primary anti-Myc and rabbit polyclonal anti-calnexin were and with in The secondary and were in the an analysis of was done using the Cell cells were three with containing Cells were at with of for To confirm that the not intracellular proteins, the was in in one was with the chicken anti-Panx1 and the with the mouse anti-α-tubulin In with and cells a rPanx1 Kim PubMed Scopus Google or were for at 37 with or in and or was by with at 37 in Cells were for several in the presence of The newly proteins were at the of the time with the was for the cells with or cells were with microscopy or in for of and cells were in and for on with to and were as G. Kim J. O. PubMed Scopus Google Scholar). and or were on The for these and Kim Cell Res. PubMed Scopus Google Scholar); were for channel was as G. D. A Scholar). The was from oocytes by The oocytes were with in Panx1 or and for The membrane was with and the oocytes was with the D.C. Kim J. PubMed Scopus Google Scholar) at time Pannexin1 a state of a protein can be analyzed using as it been shown for connexins Kim 2000; PubMed Scopus Google Scholar). To Panx1 forms a multimeric we treated of cells with DSP, is an a of two were observed Based on are to to the and hexameric forms kDa). a of a of was observed the kDa), kDa), kDa), and hexamer not the by in the presence of in the of the forms to the kDa). the in the absence of not reverse the and the were observed not the hexamer were not observed at of that the the formation of an and not an To confirm this the was on a that of proteins, using as shown in the the it to the Panx1 hexameric form kDa). these the hexamer and we observed the as a single Pannexin1 and at the Cell of Panx1 protein by revealed the presence of as shown in cell from or cells were using an anti-Panx1 or an anti-Myc Panx1 were observed to we to as and A pattern was observed for the protein from and of where the and were whereas in rat and in a mouse cell only the was evident that the different Panx1 were not to in of The presence of is to as it has been shown with connexins Kim J. Biol. PubMed Scopus Google or glycosylation as for channels I. Kim J. E. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). To the pattern of Panx1 could different we used an alkaline in no was observed in the pattern of the as with membrane that is not for the of To the pattern could different glycosylated a of or protein was using Endo and PNGase F, not between the N-linked glycosylated shown in Endo only the this to in the The and were Endo PNGase and that the the protein expressed on the cell surface The was not by PNGase it to the protein. were in cell where the was to PNGase and the one was not not In rPanx1 is and forms three and of Pannexin1 by Site-directed in analyzed the amino sequence of rPanx1 for putative glycosylation sites Ref. R. Kim S. Scholar). We found four predicted N-glycosylation sites at and and are in the predicted whereas Asn-254 is in the extracellular of To of the predicted sites is important as a glycosylation we used site-directed mutagenesis to intracellular and one extracellular and were to The a and this residue is at the of the it unlikely that it be a glycosylation The to were expressed in cells and examined by analysis and shown in the was to the protein as it different of protein and the was as it the and and was to PNGase not Furthermore, the was only glycosylated as it the but the The localization of wild-type, and proteins was with that of the protein Both wild-type and proteins were at the cell surface A and However, it that and were in the and with and To Panx1 was at the cell we or cells and analyzed the protein by and The proteins were by an and with the protein shown in A and and the was that it to the protein on the cell surface. the was to Endo we it to glycosylated the is found in the this is to some intracellular proteins by the as shown in the of the with an However, there is relatively little labeling of in the with the Cell surface of the confirmed the cell surface expression of the To we rPanx1 is glycosylated at residue this is an important in the trafficking of the and N-glycosylation at the Asn-254 residue induces and cell surface of and and and and proteins were with a chicken polyclonal To confirm that the not intracellular proteins, a was in and with an We the rPanx1 formed a multimeric channel. with the of to the kDa), kDa), kDa), and hexamer of rPanx1 not The hexamer and were the in this The single to the was that the mutation could with the of the protein to completely in an channel. the by in the presence of in the of the to the by the of Pannexin1 on the Cell to site-directed we the of protein glycosylation in targeting Panx1 to the plasma membrane. We used of the unit to the and N-linked cells a rPanx1 were treated with for different the of time analysis revealed a significant in the Panx1 and an in the proteins To Panx1 proteins we used the labeling with green fluorescent and fluorescent G. Kim J. O. PubMed Scopus Google Scholar). to two of cells were with for different of time in medium or in the presence of with and of the cells with a polyclonal for Panx1 confirmed the of staining not The proteins were distributed throughout the plasma membrane with the staining the gap junction was a the of and labeling was a protein the presence of with the green fluorescent was in not the proteins were to this for the proteins the cytoplasm and no labeling on the plasma membrane was Furthermore, following with the protein there was labeling in the intracellular the plasma membrane not These data confirmed the of glycosylation in targeting the newly Panx1 proteins to the cell surface. To the of the containing the proteins, were with for in with and The the of Panx1 with an important role of in Panx1 in this cell of Pannexin1 Panx1 proteins form gap junction-like structures we these proteins at using and G. Kim J. O. PubMed Scopus Google Scholar) to or A and expressed in cells. One of the of this is the to and electron The cells were with and for of of in the presence of to an electron product the proteins to be at In A and the staining with in cells the electron of cells at F, and show that Panx1 in areas of cell to cell apposition not appear as canonical gap and the intercellular between adjacent cells is with in the gap For the Panx1 is the plasma membrane whereas is not but rather in a cells the do not show product Pannexin1 studies were to Panx1 proteins were to form intercellular channels. A for gap junction is the Kim Cell Res. PubMed Scopus Google Scholar). We these in cells that expressed Panx1 or in cells or a rPanx1 was directly for to protein expression. shown in of place in cells but not in Panx1, or or A and for gap junction is the in Xenopus oocytes G. Kim R. E. C. J. Full Text PDF PubMed Scopus Google Scholar). were with Panx1 and of in and was by at and oocytes as and oocytes as was as expressed in forms gap junction channels and in the membrane. for an of the of Panx1 and protein expression in the membrane by the membrane that formed gap junctions was and with The junctional in Panx1 on the was at the Panx1 to form a significant of gap junction channels expression as by the Panx1-specific membrane Based on the membrane and the of and Panx1 channels, of were in the form of gap junction channels, whereas of were in the form of and junctional of pairs Panx1 or D.C. Kim J. PubMed Scopus Google (13Verselis V.K. Kim E.B. Bukauskas F.F. Braz. J. Med. Biol. Res. 2000; 33: 379-389Crossref PubMed Scopus (41) Google (12Dvoriantchikova G. Kim D. Panchin Y. Shestopalov V.I. FEBS Lett. 2006; 580: 2178-2182Crossref PubMed Scopus (84) Google S. Kim E. Qiu F. Spray D.C. Dahl G. FEBS Lett. 2007; 581: 483-488Crossref PubMed Scopus (353) Google in a new The for and Pannexin1 by a of of were the protein in the plasma membrane might by the the of gap junction channel formation is of the G. Kim R. E. C. J. Full Text PDF PubMed Scopus Google the observed in the to form gap junction channels in could be to this To gap junction formation at the was at a and the oocytes were paired The to as by from to were and for time and that the gap junction formation of this by that of the by In with Xenopus oocytes with the of cells only a membrane and junctional (0.5 to the expression and a of The oocytes a membrane oocytes a mechanism of trafficking to a of The data presented here show for the first time that Panx1 forms a multimeric channel that is on the extracellular at on that Panx1 forms a hexameric channel to a connexin we observed several that on to the and hexamer of the hexamer were not observed at a of that the the formation of an rather an The and of to proteins in the of the endoplasmic and in the in different shown here that rPanx1 is and three different forms are the protein that is not by the the in the that is to Endo and the protein that is expressed on the cell surface. only at sites in the of the that to the extracellular of the protein. studies that the Asn-254 is to the extracellular of the protein the predicted transmembrane of In with these microscopy on cells Panx1 proteins a at the carboxyl that the was to the only the cells been not These the by and (3Locovei S. Kim L. Dahl G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7655-7659Crossref PubMed Scopus (421) Google Scholar) in an to Xenopus oocytes staining on the cell whereas the oocytes only of three revealed that trafficking to the cell surface is by two of these three confirmed that only the is whereas is These that glycosylation at Asn-254 is important for plasma membrane targeting of the Panx1 protein. Furthermore, N-glycosylation by in an of the Panx1 proteins the cytoplasm with no labeling on the plasma membrane. the was only as by the presence of the and its trafficking to the cell surface was We propose that may be to the but with proteins with the or may its glycosylation to the plasma membrane. The to proteins is a in of the of We used the labeling with green fluorescent and fluorescent to two of and proteins to be with each at the plasma membrane. Panx1 proteins for were to the of this for Panx1 staining the gap junction was observed as confirmed by of and Panx1 proteins in and Furthermore, the of the Panx1 proteins confirmed their on the cell surface. plasma the intercellular between adjacent cells was the of to the extracellular of Panx1 the apposition to a between two hemichannels the into a gap of glycosylation sites into connexins these gap proteins G. Kim R. J. Full Text PDF PubMed Scopus Google Scholar). Furthermore, show that these oligomers do not between cells as has been shown for connexins. The presence of a glycosylation site at the extracellular the on the plasma membrane of Panx1, and the of that pannexins form a new class of channel proteins from connexins, studies in by Dahl and (3Locovei S. Kim L. Dahl G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7655-7659Crossref PubMed Scopus (421) Google Scholar) and in cells, and neurons by and Y. Kim J.B. Abrams C.K. Scherer S.S. Glia. 2007; 55: 46-56Crossref PubMed Scopus (148) Google Scholar). A by et al. Kim Bruzzone R. Res. 2007; PubMed Scopus Google Scholar) in cells transfection with or These cells do not connexins or pannexins; from these cells are do connexins and the plasma it is that the Panx1 of connexins and cells connexin intercellular channels rather putative intercellular channels. In paired Panx1 expression in the formation of junctional only with cell to cell of and is in with the by Bruzzone et al. (2Bruzzone R. Kim S.G. Barbe M.T. Herb A. Monyer H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13644-13649Crossref PubMed Scopus (614) Google Scholar) where were used to the of Panx1 to form gap junction channels. of junctional from an of no significant gap junction function to Panx1 could be a of cell to cell However, Panx1 was for a membrane expression of the protein. In gap junction channels A junctional was and gap junction channels and were the of cells and their the for in the of for Panx1 to form gap junction channels with a role in a in cells in tissues where might be In the expression the form of Panx1 a of the protein (3Locovei S. Kim L. Dahl G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7655-7659Crossref PubMed Scopus (421) Google Scholar). glycosylation in these cells is Kim J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. Kim D. R. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Kim PubMed Scopus Google Scholar). It is that the trafficking some or glycosylated Panx1 reaches the plasma membrane and can be used for gap junction channel this may be an of such a Furthermore, the in pairs can be an of an the of gap junction formation in paired oocytes (2Bruzzone R. Kim S.G. Barbe M.T. Herb A. Monyer H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13644-13649Crossref PubMed Scopus (614) Google Scholar) could be a of the expression on The formation of gap junction channels in this may not the in of the pannexin protein. In that rPanx1 cell surface expression can be by such as a and a Pannexins are expressed in many different tissues, and the cell surface expression in different locations. In and cells, for example, the expression pattern of Panx1 to be and only the to the protein is observed by of membrane proteins can their folding, and We propose that N-glycosylation of Panx1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface and their function in different tissues. We are to Dr. Roberto Bruzzone for the cDNA for the and We for with site-directed and for with the cell and

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