Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

Abstract

Pannexins (Panx) are a class of integral membrane proteins that have been proposed to exhibit characteristics similar to those of connexin family members. In this study, we utilized Cx43-positive BICR-M1R k cells to stably express Panx1, Panx3, or Panx1-green fluorescent protein (GFP) to assess their trafficking, cell surface dynamics, and interplay with the cytoskeletal network. Expression of a Sar1 dominant negative mutant revealed that endoplasmic reticulum to Golgi transport of Panx1 and Panx3 was mediated via COPII-dependent vesicles. Distinct from Cx43-GFP, fluorescence recovery after photobleaching studies revealed that both Panx1-GFP and Panx3-GFP remained highly mobile at the cell surface. Unlike Cx43, Panx1-GFP exhibited no detectable interrelationship with microtubules. Conversely, cytochalasin B-induced disruption of microfilaments caused a severe loss of cell surface Panx1-GFP, a reduction in the recoverable fraction of Panx1-GFP that remained at the cell surface, and a decrease in Panx1-GFP vesicular transport. Furthermore, co-immunoprecipitation and cosedimentation assays revealed actin as a novel binding partner of Panx1. Collectively, we conclude that although Panx1 and Panx3 share a common endoplasmic reticulum to Golgi secretory pathway to Cx43, their ultimate cell surface residency appears to be independent of cell contacts and the need for intact microtubules. Importantly, Panx1 has an interaction with actin microfilaments that regulates its cell surface localization and mobility.The pannexin family is a new class of integral membrane glycoproteins that have been identified to share sequence homology with the invertebrate gap junction proteins, innexins (1). Unlike the connexin family that is composed of 21 members (2), both the human and mouse genomes contain only three pannexin-encoding genes (Panx1, Panx2, and Panx3) (1). Although connexins and pannexins exhibit no sequence homology, pannexins are predicted to share similar topology with connexins, which includes four transmembrane domains, two extracellular loops, a cytoplasmic loop, and intracellular amino and carboxyl termini (3-5). Our previous study has shown that ectopically expressed Panx1 and Panx3 are capable of trafficking to normal rat kidney (NRK) 2 cell surfaces; however, their distribution profile at cell-cell interfaces is not typically clustered or punctate as seen for Cx43 (5). Consistently, electron micrographs of Panx1 overexpressing Madin-Darby canine kidney cells also revealed dispersed Panx1 localization at the plasma membrane with no evidence of gap junction plaques (4).Cx43 has a relatively short half-life of ϳ1-3 h (6). As a result, Cx43 subunits assembled into connexons within the transGolgi network (7) are constantly being transported to and removed from the plasma membrane (8). Recent reports provide evidence that although Panx1 appears to form similar hexameric channel units defined as pannexons (4), they exhibit slower turnover dynamics in comparison with Cx43, as assessed by the use of pharmacological blocke...

The cellular life of pannexins

“…39 Pannexins do not appear to form the kind of close channel packing that would lead to dense crystalline-like structures, as reported for Cx43. 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. Consistent with this latter finding, Panx1 was found to bind directly to actin, which could suggest that microfilaments act to stabilize the cell surface distribution of Panx1 or even participate in Panx1 delivery or internalization.…”

“…9,19,38 We have shown that Panx1 and Panx3 follow a classical ER-Golgi secretory pathway in a Sar1-mediated COPII-dependent manner. 39 Once in the Golgi apparatus pannexins can undergo further editing and be modified into complex glycoproteins that are preferentially targeted to the plasma membrane as was evidenced by cell surface biotinylation assays 9 (Figure 4). Interestingly, the Gly0, Gly1, and Gly2 isoforms of Panx1 and Panx3 can all be trafficked to the cell surface to some degree, suggesting that glycosylation is not absolutely required for them to reach the cell surface albeit it almost assuredly enhances the efficiency of the process.…”

“…Consistent with this latter finding, Panx1 was found to bind directly to actin, which could suggest that microfilaments act to stabilize the cell surface distribution of Panx1 or even participate in Panx1 delivery or internalization. 39 …”

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