Boassa Daniela, Nguyen Phuong, Hu Junru, Ellisman Mark H, Sosinsky Gina E
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA.
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA ; Department of Neurosciences, University of California San Diego, La Jolla, CA, USA.
Front Cell Neurosci. 2015 Feb 2;8:468. doi: 10.3389/fncel.2014.00468. eCollection 2014.
Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.
泛连接蛋白2（Panx2）是泛连接蛋白家族三个成员中最大的一个。泛连接蛋白在拓扑结构上与连接蛋白和内向整流离子通道蛋白相关，但它们的功能不同于形成间隙连接。我们之前表明，泛连接蛋白形成寡聚通道，但与连接蛋白和内向整流离子通道蛋白不同的是，它们只形成单膜通道。中枢神经系统（CNS）中已记录到高水平的Panx2信使核糖核酸（mRNA）和蛋白质。虽然泛连接蛋白1（Panx1）相当普遍，泛连接蛋白3（Panx3）存在于皮肤和结缔组织中，但两者都完全糖基化，运输到质膜，并且具有与细胞外三磷酸腺苷（ATP）释放相关的功能。在这里，我们描述了外源性Panx2和Panx1蛋白在犬肾上皮细胞（MDCK）、人宫颈癌细胞（HeLa）和人胚肾细胞（HEK 293T）中的运输和亚细胞定位，以及中枢神经系统中内源性Panx1和Panx2的模式。发现Panx2存在于细胞内定位，部分进行了N-糖基化，并且其定位与Panx1不重叠。对海马切片进行免疫标记，以检测星形胶质细胞蛋白胶质纤维酸性蛋白（GFAP）、Panx1和Panx2，共聚焦图像显示，Panx1和Panx2这两种异构体在星形胶质细胞和神经元的不同亚细胞区室中定位。使用为相关光镜和电镜开发的带有附加基因标签的Panx2重组融合体，然后在不同细胞系中表达，我们确定Panx2定位于细胞内囊泡的膜上，而不是像钙连蛋白共定位实验最初表明的那样定位于内质网中。用针对特定囊泡区室的蛋白质标记物进行双免疫荧光成像显示，Panx2囊泡起源于早期内体。在电子断层扫描体积中，这些囊泡的横截面显示出精细的结构细节，并且与肌动蛋白丝紧密相邻。因此，在不同亚细胞区室表达的泛连接蛋白可能发挥不同的功能作用，特别是在神经系统中。
