Pathways Regulating the Trafficking and Turnover of Pannexin1 Protein and the Role of the C-terminal Domain

Abstract

Pannexin1 (Panx1) is an integral membrane protein comprised of three species as follows: an unglycosylated core-Gly0, a high mannose-Gly1, and a complex glycosylated Gly2 species. Although Panx1 channels mediate several cellular responses, the domain regulating its oligomerization and cell surface trafficking and the mechanisms governing its internalization and degradation have not been identified. This study characterizes the role of the Panx1 C-tail domain by truncating the polypeptide at residue 307 and expressing the mutant in BICR-M1R k and HEK-293T cells. Enzymatic digestion and immunolabeling assays revealed that the Panx1 T307 -RFP was glycosylated primarily to the high mannose species consistent with its retention in the endoplasmic reticulum. Co-expression of Panx1 T307 -RFP with Panx1 followed by co-immunoprecipitation assays revealed that the mutant and Panx1 could interact, whereas biotinylation assays showed that this interaction inhibited Panx1 from maturing into the Gly2 species and reaching the cell surface. Additional inhibitor studies indicated that the degradation of the mutant was via proteasomes, whereas Panx1 was degraded by lysosomes. Analysis of the pathways important in Panx1 internalization revealed partial co-distribution of Panx1 with many molecular constituents of the endocytic machinery that include clathrin, AP2, dynamin II, caveolin-1, and caveolin-2. However, co-immunoprecipitation assays together with the disruption of lipid rafts by methyl-␤-cyclodextrin suggest that Panx1 does not engage this endocytic machinery. Furthermore, dominant-negative and pharmacological studies revealed that Panx1 internalization was dynamin II-independent. Collectively, these results indicate that the oligomerization and trafficking of Panx1 are regulated by the C-terminal domain, whereas internalization of long lived Panx1 channels occurs in a manner that is distinct from classical endocytic pathways.Pannexins were originally discovered due to their shared sequence homology with the invertebrate gap junction proteins, innexins (1). The pannexin family is comprised of three members as follows: Panx1, Panx2, and Panx3 (1). Although the scope of physiological significance of pannexin family members is only beginning to emerge, it has been reported that Panx1 holds importance in forming conduits for ATP release (2-4), propagation of Ca 2ϩ waves (5), and neuronal and immunological inflammasome (6 -8). Additionally, activation of Panx1 channels has been implicated in ischemic cell death (9) and seizure-like activities (10). Although Panx1 single channel conductances of 550 picosiemens (2) have been correlated with ionic dysregulation during ischemic conditions, subsequently leading to neuronal necrosis (9), the N-methyl-D-aspartate receptor-induced opening of Panx1 channels has been implicated in hippocampal epileptiform seizure-like activity (10). Other studies have associated Panx1 channel activation with apoptosis of Xenopus oocytes by forming a pore unit with the death complex of the P2X7 receptor (...

Analysis of a pannexin 2-pannexin 1 chimeric protein supports divergent roles for pannexin C-termini in cellular localization

“…The current study lends further support to a growing body of work indicating that the Panx1 C-terminus is important for traffi cking (Gehi et al, 2011). The localization of chimeric Panx2 with the C-terminus of Panx1 was distinct from wildtype Panx2 expression, suggesting that elements within the Panx2 C-terminus are critical for its proper localization and are absent from the Panx1 C-terminus.…”

“…One of the more recently emerging vital structural components in traffi cking of Panx1 is its C-terminus (Gehi et al, 2011). This region is quite dissimilar between Panx1 and Panx2.…”

“…Panx1CT -EGFP plasmid, or EGFP control plasmid (Gehi et al, 2011;Penuela et al, 2007). Where indicated, cells were stained with tetramethylrhodamine isothiocyanate conjugated wheat-germ agglutinin (WGA-TRITC; Molecular Probes/Life Technologies, Burlington, Ontario, Canada) according to the manufacturer ' s protocol, before fi xing with 3.7% formaldehyde in PBS for 15 min.…”

The cellular life of pannexins

“…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.…”

“…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. 41 Therefore, Panx1 internalization may be governed by molecular machinery that could potentially involve actin microfilaments. 39 …”