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

Abstract

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), 2 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 (1). 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 (NH 2 ) 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 seconda...

The cellular life of pannexins

“…9,39,45,46 These same three subcellular localization profiles have also been identified for endogenously expressed Panx1 in a variety of cells and tissues (Figures 6 and 7) that include: melanoma cells, 39 MDCK cells, 29 human facial epidermis, 26 rat atrial and ventricular primary cardiomyocytes, 47 Hensen, Claudius, and Boettcher cells of the rat cochlea, 46 a mouse osteoblast cell line (MC3T3-E1), 45 spiral limbus, 46 and in the murine spleen. 26 It is important to note that in order to elucidate the diverse localization profiles of pannexins it is imperative to validate the specificity of the antibodies being used.…”

“…26 It remains to be discovered if phosphorylation plays a regulatory role on trafficking or channel function of pannexins as is the case for connexins. 28 On the other hand, the predicted extracellular loop sequence consensus sites for N-glycosylation ( Figure 1) in Panx1 (N254) and Panx3 (N71) have been confirmed to indeed be sites of glycosylation as demonstrated by site-directed mutagenesis studies 9,26,29 and by sensitivity to N-glycosidase F and endoglycosidase H enzymatic digestion. These complementary approaches led to the conclusion that there were two levels of Panx1 and Panx3 glycosylation and three distinct species: the non-glycosylated core species-Gly0, the predominant endoplasmic reticulum (ER) resident high-mannose species-Gly1, and the complex glycosylated species-Gly2 ( Figure 3).…”

“…Once Panx1 is destined to internalize, our own studies using inhibitors of lysosomal function 41 as well as other studies that showed colocalization of Panx1 with lysotracker 29 suggest that Panx1 is destined for ultimate lysosomal degradation (Figure 4). However, the pathways involved in classical endocytic internalization, including clathrin, dynamin II, and caveolins-1 and -2, do not appear to be engaged in the internalization of Panx1.…”

“…40 Electron micrographs revealed that Panx1 was localized uniformly at the cell surface in single membranes that were at least 20-50 nm from an apposing cell membrane. 29 Nocodazole-induced disruption of microtubules did not significantly alter the cell surface distribution of Panx1 and Panx3. 39 On the other hand, disruption of microfilaments revealed concomitant intracellular accumulation of Panx1 and Panx3 leaving only a subpopulation at the cell surface.…”

“…In addition, Panx2 also encodes a potential N-linked glycosylation consensus site in its first EL domain at asparagine 86 ( Figure 1) and is sensitive to deglycosylating enzymes, but glycosylation of the N86 residue has yet to be confirmed by a site-directed mutagenesis study. 9 N-glycosylation of pannexins seems to play a key role in regulating their traffic to the cell surface, 29 as the Gly2 complex forms are more abundant at the plasma membrane, whereas both the Gly0 and Gly1 are preferentially found as residents of the ER. 9 Glycosylation also plays a role in regulating the potential intermixing of pannexin family members into channels because the Gly2 glycosylation species of Panx1 appears incapable of being coimmunoprecipitated (coIP) with Panx2 when coexpressed in HEK293T cells.…”