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

Abstract

Pannexins (Panxs) are a three-member family of large pore ion channels permeable to ions and small molecules. Recent elegant work has demonstrated that the Panx1 C-terminus plays an important role in channel traffi cking. Panx2, another family member, has a longer and highly dissimilar C-terminus. Interestingly, Panx1 is readily found at the plasma membrane, while Panx2 is mainly present on intracellular membranes. Here we used overlap-extension cloning to create the fi rst chimeric Panx, consisting of Panx2 with the Panx1 C-terminus (Panx2 Panx1CT ), to determine whether the Panx1 C-terminus infl uences the traffi cking of Panx2. We are the fi rst to observe a high level of co-localization between Panx2 and the endolysosomal enriched mannose-6-phosphate receptor. Interestingly this distinct localization of Panx2 is altered by the presence of the Panx1 C-terminus. These novel observations support previous data indicating the importance of the C-terminus in the control of Panx traffi cking, and highlight the complexity of molecular signals involved.

Novel Variant in the ANK2 Membrane-Binding Domain Is Associated With Ankyrin-B Syndrome and Structural Heart Disease in a First Nations Population With a High Rate of Long QT Syndrome

“…All plasmids were verified by sequencing prior to use. Cell lysis was performed as previously described 17–19 . Lysates were heated for 5 minutes (95°C) under reducing conditions, separated by SDS-PAGE and transferred to 0.2 µm pore-size polyvinyldene fluoride (PVDF) membrane for 3 hours at 200mA.…”

Pore positioning

“…Interestingly, we have uncovered signal sequences for clathrin-mediated endocytosis and endolysosomal localization in each Panx. Moreover, using live cell confocal microscopy of Panx1-EGFP expressing Neuro-2A neuroblastoma cells (which endogenously express Panx1) 17 stimulated with increased extracellular potassium, we observe EGFP-positive vesicles undergoing retrograde transit from the plasma membrane to subcellular regions (Fig. 3a).…”

“…In addition to Panxs at the cell surface, which have dominated functional studies, Panxs are also detected in intracellular compartments of diverse cell types, including neural cells 17,18 keratinocytes, 16,19 prostate adenocarcinoma cells, 20 chondrocytes, 14 and differentiated osteoblasts. 15 In contrast with Panx1 and Panx3, which are readily found at the plasma membrane, the contrarian Panx2 is actually primarily localized to intracellular compartments, with limited plasma membrane expression having been noted only in overexpression systems 21 and some neuronal cell types.…”

“…17 In neuroblastoma cells, the chimeric Panx protein exhibits diffuse intracellular localization compared with the predominantly plasma membrane expression of Panx1, and the endolysosomal localization of Panx2 (Fig. 1B).…”

Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology?

“…Panx2 on the other hand has been proposed to form octameric channels [60]. However, since Panx2 is largely found to be present in intracellular compartments, Panx1 is suggested to be the main constituent of the Panx channel in the CNS [28,61,62]. In addition, Panx channels do not appear to form GJCs, as docking is impaired due to the presence of glycosylation patterns on the extracellular loops ( Fig.…”