Parietal cells, which secrete gastric acid, and the apical surface of epithelial cells of the belly were strongly reactive for Panx2 (Physique ?(Figure5A).5A). qPCR, Western blotting and immunofluorescence. Panx2 protein levels were readily detected in every tissue examined, even when transcriptional analysis predicted very low Panx2 protein expression. Furthermore, our results indicate that Panx2 transcriptional activity is usually a poor predictor of Panx2 protein abundance and does not correlate with Panx2 protein levels. Despite showing disproportionately high transcript levels, the CNS expressed less Panx2 protein than Alagebrium Chloride any other tissues analyzed. Additionally, we showed that Panx2 protein does not localize at the plasma membrane like other space junction proteins but remains confined within cytoplasmic compartments. Overall, our results demonstrate that this endogenous expression of Panx2 protein is not restricted to the CNS and is more ubiquitous than in the beginning predicted. Keywords: pannexin 2, space junction, gene transcription, protein expression, protein distribution, central nervous system (CNS), mouse, mRNA Introduction Space junction proteins are traditionally described as aqueous plasma membrane channels which allow quick cell-to-cell communication by directly connecting the cytoplasm of Alagebrium Chloride adjacent cells. In chordates, connexins (Cxs) are the canonical space junction proteins while space junctions in invertebrates are created exclusively by the evolutionarily unrelated innexin (Inx) family. In 2000, another small gene family named pannexin (Panx) was recognized based LFNG antibody on sequence homology with the Inx family and was found to be expressed alongside Cxs in chordates (Panchin et al., 2000). Three unique Panx paralogs (Panx1, Panx2 and Panx3) were initially recognized in vertebrates (Panchin et al., 2000; Panchin, 2005; Barbe, 2006) but recent studies showed that Panx1 has been retained as two impartial ohnologs Alagebrium Chloride in teleost as a result of an ancestral whole genome duplication (Bond et al., 2012; Kurtenbach et al., 2013). Despite the lack of sequence similarity between Inxs/Panxs and Cxs, both families share structural resemblance. Cxs and Panxs both have a predicted topology consisting of four membrane-spanning domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-termini (Panchin, 2005). Despite sharing structural resemblance with Cxs, the ability of Panx channels to form space junctional coupling remains controversial. A few groups reported that Panx1 and Panx3 can form cell-cell junctional channels (Bruzzone et al., 2003; Vanden Abeele et al., 2006; Lai et al., 2007; Ishikawa et al., 2011; Sahu et al., 2014) but their observations were limited primarily to heterologous or over-expression systems and undisputable evidence supporting Panx-based coupling is still lacking. In contrast to Cxs, all three Panxs are glycosylated at their extracellular loops (Penuela et al., 2009) with carbohydrate moieties that sterically hinder the docking of channels from adjacent cells (Boassa et al., 2007). Therefore, it is largely accepted that under physiological conditions, Panx channels primarily form non-junctional membrane channels controlling the exchange of ions and small molecules between the cytoplasm and extracellular space and do not significantly contribute to direct cell-to-cell space junctional communication (Sosinsky et al., 2011). Several gene expression profiling studies reported that Panx2 transcriptional activity is largely restricted to the central nervous system in human (Baranova et al., 2004), rat (Bruzzone et al., 2003) and zebrafish (Zoidl et al., 2008; Bond et al., 2012). Minimal Panx2 mRNA levels have also been detected in some non-neural tissues such as the vision, thyroid, prostate, kidney, liver, heart and olfactory epithelium (Bruzzone et al., 2003; Dvoriantchikova et al., 2006; Bond et al., 2012; Zhang et al., 2012) but given the much larger Panx2 mRNA levels found in the CNS, Panx2 transcript and corresponding protein are largely assumed to be primarily expressed in the CNS. In the healthy brain, Panx2 protein was shown to have Alagebrium Chloride a complex distribution pattern and is expressed in pyramidal cells and interneurons alike (Zappal et al., 2007). Interestingly, Panx2 protein was also detected in astrocytes following ischemia in the rat but not in the healthy brain (Zappal et al., 2007). Panx2 protein is also present in hippocampal neural progenitors and mature neurons both and (Swayne et al., 2010). However, because Panx2 is usually believed to be primarily CNS-specific, the mapping of Panx2 protein distribution in other tissues has not been undertaken. In this study, we compared Panx2 gene transcription and protein expression profiles in mouse tissues using a combination of real-time qPCR, Western blot and immunofluorescence. Our results reveal that Panx2 mRNA and protein levels are not correlated and demonstrate that Panx2 protein expression is more ubiquitous than in the beginning predicted. Materials and methods Animal care All experiments were performed in accordance with the guidelines established by the Canadian Council on Animal Care and were.