Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
J Biol Chem. 2010 Mar 19;285(12):9147-60. doi: 10.1074/jbc.M109.082008. Epub 2010 Jan 10.
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 co-sedimentation 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.
缝隙连接蛋白（Panx）是一类整合膜蛋白，被认为具有类似于连接蛋白家族成员的特征。在这项研究中，我们利用 Cx43 阳性的 BICR-M1R(k) 细胞稳定表达 Panx1、Panx3 或 Panx1-绿色荧光蛋白（GFP），以评估它们的转运、细胞表面动力学以及与细胞骨架网络的相互作用。Sar1 显性失活突变体的表达表明，Panx1 和 Panx3 的内质网到高尔基体的运输是通过 COPII 依赖性囊泡介导的。与 Cx43-GFP 不同，光漂白后荧光恢复研究表明，Panx1-GFP 和 Panx3-GFP 在细胞表面均保持高度流动性。与 Cx43 不同，Panx1-GFP 与微管没有可检测到的相互关系。相反，细胞松弛素 B 诱导的微丝破坏导致细胞表面 Panx1-GFP 严重丢失，留在细胞表面的 Panx1-GFP 可恢复部分减少，Panx1-GFP 囊泡运输减少。此外，共免疫沉淀和共沉淀测定显示肌动蛋白是 Panx1 的一种新的结合伴侣。总的来说，我们得出结论，尽管 Panx1 和 Panx3 与 Cx43 共享一个共同的内质网到高尔基体分泌途径，但它们最终的细胞表面驻留似乎独立于细胞接触和完整的微管。重要的是，Panx1 与肌动蛋白微丝相互作用，调节其细胞表面定位和流动性。
