Cx31.1 can selectively intermix with co-expressed connexins to facilitate its assembly into gap junctions.

Connexins are channel-forming proteins that function to facilitate gap junctional intercellular communication. Here, we use dual cell voltage clamp and dye transfer studies to corroborate past findings showing that Cx31.1 (encoded by GJB5) is defective in gap junction channel formation, illustrating that Cx31.1 alone does not form functional gap junction channels in connexin-deficient mammalian cells. Rather Cx31.1 transiently localizes to the secretory pathway with a subpopulation reaching the cell surface, which is rarely seen in puncta reminiscent of gap junctions. Intracellular retained Cx31.1 was subject to degradation as Cx31.1 accumulated in the presence of proteasomal inhibition, had a faster turnover when Cx43 was present and ultimately reached lysosomes. Although intracellularly retained Cx31.1 was found to interact with Cx43, this interaction did not rescue its delivery to the cell surface. Conversely, the co-expression of Cx31 dramatically rescued the assembly of Cx31.1 into gap junctions where gap junction-mediated dye transfer was enhanced. Collectively, our results indicate that the localization and functional status of Cx31.1 is altered through selective interplay with co-expressed connexins, perhaps suggesting Cx31.1 is a key regulator of intercellular signaling in keratinocytes.

Global pannexin 1 deletion increases tumor-infiltrating lymphocytes in the BRAF/Pten mouse melanoma model.

Immunotherapies for malignant melanoma seek to boost the anti-tumoral response of CD8+ T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel-forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1-/-) mice with the inducible melanoma model BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten-driven primary tumor formation or improve survival. However, tumors in BPC-Panx1-/- mice exhibited a significant increase in the infiltration of CD8+ T lymphocytes, with no changes in the expression of early T-cell activation marker CD69, lymphocyte activation gene 3 protein (LAG-3) checkpoint receptor, or programmed cell death ligand-1 (PD-L1) in tumors when compared to the BPC-Panx1+/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF/Pten-driven melanoma progression in vivo, it does increase the infiltration of effector immune T-cell populations in the tumor microenvironment. We propose that PANX1-targeted therapy could be explored as a strategy to increase tumor-infiltrating lymphocytes to boost anti-tumor immunity.

Pattern of Visual-Motor Integration, Visual Perception, and Fine Motor Coordination Abilities in Children Being Assessed for Fetal Alcohol Spectrum Disorder.

OBJECTIVE: Motor skill assessment is part of the fetal alcohol spectrum disorder (FASD) multidisciplinary assessment. Some clinicians opt to exclude assessment of the subcomponents of visual-motor integration (visual perception and motor coordination), on the assumption that challenges will be revealed based on the assessment of visual-motor integration. The objective is to describe the visual-motor integration, visual perception, and fine motor coordination pattern of abilities in children with confirmed prenatal alcohol exposure being assessed for fetal alcohol spectrum disorder. METHODS: This cross-sectional study included 91 children (65 males; mean age: 10 years, 6 months SD = 2 years, 10 months) undergoing assessment for FASD. Friedman and Wilcoxon statistics were used to compare mean visual-motor integration, visual perception, and fine motor coordination percentiles from the Beery-Buktenica Developmental Test of Visual-Motor Integration, Sixth Edition (Beery-6). RESULTS: Children being assessed for FASD (n = 91) had the highest normative scores in visual perception, followed by visual-motor integration and fine motor coordination (mean percentiles (SD): 35.9 (24.9), 20.6 (18.3), and 13.8 (15.5), respectively) (χ 2 distribution = 46.909, p ≤ 0.001). CONCLUSION: Children being assessed for FASD experience more challenges with fine motor coordination compared with visual-motor integration and visual perception tasks. This pattern differs from the pattern established for the general population in which tasks that require visual-motor integration are more challenging than tasks that isolate visual perception and fine motor coordination. These results suggest that fine motor coordination should be included in FASD diagnostic assessments and considered as an area for intervention.

Expression of pannexin1 in lung cancer brain metastasis and immune microenvironment.

Brain metastases are the most common central nervous system malignancy, and the leading cause of cancer-related deaths. Non-small cell lung carcinomas (NSCLC) comprise the most common cell of origin. Immunotherapy, particularly checkpoint inhibitors, has emerged as the standard of care for many patients with advanced lung cancer. Pannexin1 (PANX1) is a transmembrane glycoprotein that forms large-pore channels and has been reported to promote cancer metastasis. However, the roles of PANX1 in lung cancer brain metastases and tumor immune microenvironment have not been characterized. 42 patient-matched formalin-fixed paraffin-embedded tissue samples from lung carcinomas and the subsequent brain metastases were constructed into three tissue microarrays (TMAs). PANX1 and markers of tumor-infiltrating immune cells (CD3, CD4, CD8, CD68, and TMEM119) were assessed using immunohistochemistry and digital image analysis. The expression of PANX1 was significantly higher in brain metastases than in their paired primary lung carcinoma. The high levels of PANX1 in lung carcinoma cells in the brain inversely correlated with infiltration of peripheral blood-derived macrophages. Our findings highlight the role of PANX1 in the progression of metastatic NSCLC, and the potential therapeutic approach of targeting PANX1 enhances the efficacy of immune checkpoint inhibitors in brain metastasis.

GJB4 variants linked to skin disease exhibit a trafficking deficiency en route to gap junction formation that can be restored by co-expression of select connexins.

Epidermal keratinocytes are enriched with at least nine connexins that are key regulators of epidermal homeostasis. The role of Cx30.3 in keratinocytes and epidermal health became evident when fourteen autosomal dominant mutations in the Cx30.3-encoding GJB4 gene were linked to a rare and incurable skin disorder called erythrokeratodermia variabilis et progressiva (EKVP). While these variants are linked to EKVP, they remain largely uncharacterized hindering therapeutic options. In this study, we characterize the expression and functional status of three EKVP-linked Cx30.3 mutants (G12D, T85P, and F189Y) in tissue-relevant and differentiation-competent rat epidermal keratinocytes. We found that GFP-tagged Cx30.3 mutants were non-functional likely due to their impaired trafficking and primary entrapment within the endoplasmic reticulum (ER). However, all mutants failed to increase BiP/GRP78 levels suggesting they were not inducing an unfolded protein response. FLAG-tagged Cx30.3 mutants were also trafficking impaired yet occasionally exhibited some capacity to assemble into gap junctions. The pathological impact of these mutants may extend beyond their trafficking deficiencies as keratinocytes expressing FLAG-tagged Cx30.3 mutants exhibited increased propidium iodide uptake in the absence of divalent cations. Attempts to rescue the delivery of trafficking impaired GFP-tagged Cx30.3 mutants into gap junctions by chemical chaperone treatment were ineffective. However, co-expression of wild type Cx30.3 greatly enhanced the assembly of Cx30.3 mutants into gap junctions, although endogenous levels of Cx30.3 do not appear to prevent the skin pathology found in patients harboring these autosomal dominant mutations. In addition, a spectrum of connexin isoforms (Cx26, Cx30, and Cx43) exhibited the differential ability to trans-dominantly rescue the assembly of GFP-tagged Cx30.3 mutants into gap junctions suggesting a broad range of connexins found in keratinocytes may favourably interact with Cx30.3 mutants. We conclude that selective upregulation of compatible wild type connexins in keratinocytes may have potential therapeutic value in rescuing epidermal defects invoked by Cx30.3 EKVP-linked mutants.

PANX3 Channels Regulate Architecture, Adhesion, Barrier Function, and Inflammation in the Skin.

The channel-forming glycoprotein PANX3 functions in cutaneous wound healing and keratinocyte differentiation, but its role in maintaining skin homeostasis through aging is not yet understood. We found that PANX3 is absent in newborn skin but becomes upregulated with age. We characterized the skin of global Panx3-knockout (KO) mice and found that KO dorsal skin showed sex differences at different ages but generally had reduced dermal and hypodermal areas compared with age-matched controls. Transcriptomic analysis of the KO epidermis revealed reduced E-cadherin stabilization and Wnt signaling compared with that of wild-type, consistent with the inability of primary KO keratinocytes to adhere in culture and diminished epidermal barrier function in KO mice. We also observed increased inflammatory signaling in the KO epidermis and a higher incidence of dermatitis in aged KO mice compared with that in wild-type controls. These findings suggest that during skin aging, PANX3 is critical in the maintenance of dorsal skin architecture, keratinocyte cell-cell and cell-matrix adhesion, and inflammatory skin responses.

Telotristat ethyl reverses myxomatous changes in mice mitral valves.

Rationale: Myxomatous mitral valve degeneration is a common pathological manifestation of mitral valve regurgitation, with or without valvular prolapse. In addition to similarities between naturally occurring and serotonergic valve degeneration, an increasing body of evidence has recently suggested that serotonin signaling is a regulator of degenerative valvulopathies. Studies have found that serotonin can be synthesized locally by valvular cells and serotonin receptors in turn may be activated to promote signaling. Recently, telotristat ethyl (TE) has been introduced as a treatment for carcinoid disease, by selectively inhibiting tryptophan hydroxylase 1, the rate-limiting enzyme in peripheral serotonin synthesis. TE provides a unique tool to test inhibition of serotonin synthesis in vivo, without impacting brain serotonin, to further confirm the role of local serotonin synthesis on heart valves. Objective: To confirm the link between serotonin and myxomatous valvular disease in vivo. Methods and results: A hypertension-induced myxomatous mitral valve disease mouse model was employed to test the effect of TE on valvular degeneration. Circulating serotonin and local serotonin in valve tissues were tested by enzyme immunoassay and immunohistochemistry, respectively. TE was administrated in two modes: (1) parallel with angiotensin II (A2); (2) post A2 treatment. Myxomatous changes were successfully recapitulated in hypertensive mice, as determined by ECM remodeling, myofibroblast transformation, and serotonin signaling activation. These changes were at least partially reversed upon TE administration. Conclusion: This study provides the first evidence of TE as a potential therapeutic for myxomatous mitral disease, either used to prevent or reverse myxomatous degeneration.

Motor skills and intelligence in children with fetal alcohol spectrum disorder.

AIM: To determine the relationship between motor abilities and intelligence in children and young people with prenatal alcohol exposure (PAE) being assessed for fetal alcohol spectrum disorder (FASD). METHOD: This was a cross-sectional correlational study of children and young people with PAE being assessed for FASD. The relationship between motor abilities (Movement Assessment Battery for Children, Second Edition) and intelligence (Wechsler Intelligence Scale for Children, Fourth or Fifth Edition) was calculated using correlation and regression analyses. Attention and executive function were considered as potential confounding variables. RESULTS: The relationship between motor abilities and intelligence in 73 children and young people (48 males, 25 females; aged 6-17y, mean age 10y 5mo [SD 2y 9mo]) assessed for FASD was small and statistically non-significant (r=0.05, p=0.67). INTERPRETATION: The findings confirm that motor abilities and intelligence should be assessed separately when investigating an FASD diagnosis. Intelligence scores should not be used to estimate motor abilities, nor should they dictate when motor testing be completed. Assessing intelligence and motor domains separately will enhance diagnostic accuracy, identify the need for strategies or interventions to address functional motor skills, and further define the role of physiotherapy and occupational therapy in FASD assessment and intervention.

Pannexin 2 is expressed in murine skin and promotes UVB-induced apoptosis of keratinocytes.

Pannexins (PANX) are a family of three channel-forming membrane glycoproteins expressed in the skin. Previous studies have focused on the role of PANX1 and PANX3 in the regulation of cellular functions in skin cells while PANX2, the largest member of this protein family, has not been investigated. In the current study, we explored the temporal PANX2 expression in murine skin and found that one Panx2 splice variant (Panx2-202) tends to be more abundant at the protein level and is continuously expressed in developed skin. PANX2 was detected in the suprabasal layers of the mouse epidermis and up-regulated in an in vitro model of rat epidermal keratinocyte differentiation. Furthermore, we show that in apoptotic rat keratinocytes, upon UV light B (UVB)-induced caspase-3/7 activation, ectopically overexpressed PANX2 is cleaved in its C-terminal domain at the D416 residue without increasing the apoptotic rate measured by caspase-3/7 activation. Notably, CRISPR-Cas9 mediated genetic deletion of rat Panx2 delays but does not impair caspase-3/7 activation and cytotoxicity in UVB-irradiated keratinocytes. We propose that endogenous PANX2 expression in keratinocytes promotes cell death after UVB insult and may contribute to skin homeostasis.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

Pannexin 1 binds ß-catenin to modulate melanoma cell growth and metabolism.

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of ß-catenin, a key transcription factor in the Wnt pathway. At the protein level, ß-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of ß-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with ß-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and ß-catenin interact as a potential new component of the Wnt signaling pathway.

Pannexin 1 mutation found in melanoma tumor reduces phosphorylation, glycosylation, and trafficking of the channel-forming protein.

Pannexin 1 (PANX1) is a glycoprotein that forms large pore channels capable of passing ions and metabolites such as ATP for cellular communication. PANX1 has been implicated in many diseases including breast cancer and melanoma, where inhibition or deletion of PANX1 reduced the tumorigenic and metastatic properties of the cancer cells. We interrogated the effect of single amino acid changes in various PANX1 domains using naturally occurring variants reported in cancer patient tumors. We found that a previously reported variant (Q5H) is present in cancer cells, but was not different from the wild type (Q5) in glycosylation, trafficking, or channel function and did not affect cellular properties. We discovered that the Q5H variant is in fact the highly conserved ancestral allele of PANX1 with 89% of humans carrying at least one Q5H allele. Another mutated form Y150F, found in a melanoma patient tumor, prevented phosphorylation at Y150 as well as complex N-glycosylation while increasing intracellular localization. Sarcoma (SRC) is the predicted kinase to phosphorylate the Y150 residue, and its phosphorylation is not likely to be constitutive, but rather dynamically regulated. The Y150 phosphorylation site is the first one reported to play a role in regulating posttranslational modifications and trafficking of PANX1, with potential consequences on its large-pore channel structure and function in melanoma cells.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

Accuracy of motor assessment in the diagnosis of fetal alcohol spectrum disorder.

BACKGROUND: To evaluate the accuracy of motor assessment tools listed in Fetal alcohol spectrum disorder: a guideline for diagnosis across the lifespan (Canadian Guideline) for the purpose of fetal alcohol spectrum disorder (FASD) diagnosis. Specifically, we aimed to determine: 1) diagnostic accuracy of motor assessment tools and subtests; 2) accuracy of multiple subtests versus total scores; and 3) accuracy of alternate cut-offs. METHODS: Cross-sectional diagnostic study of 63 children aged 6-17 years. Diagnostic accuracy and alternate cut-offs were calculated for the Movement Assessment Battery for Children, 2nd edition (MABC-2), Bruininks-Oseretsky Test of Motor Proficiency, 2nd edition Short Form (BOT-2SF) and Beery-Buktenica Developmental Test of Visual Motor Integration, 6th edition (BeeryVMI-6). RESULTS: The MABC-2 total motor score was more sensitive (0.30; 95% CI 0.17-0.46; p < 0.01) to motor impairment in the presence of FASD than the BOT-2SF (0.02; 95% CI 0.00-0.12) at the 2nd percentile (-2SD). The MABC-2 total motor score was more accurate than any combination of subtest scores. The Motor Coordination subtest of the BeeryVMI-6 (BeeryMC) at the 5th percentile (- 1.5SD) (sensitivity 0.68, specificity 0.90) was the most accurate subtest. CONCLUSIONS: The BOT-2SF was an inaccurate assessment tool for FASD diagnosis. The MABC-2 total motor score was the most accurate using current guidelines, though its sensitivity was still low. Further investigation into inclusion of single subtests and/or using a less conservative cut-off in the Canadian Guideline is warranted.

Targeting FER Kinase Inhibits Melanoma Growth and Metastasis.

Melanoma is one of the most aggressive types of tumors and exhibits high metastatic potential. Fes-related (FER) kinase is a non-receptor tyrosine kinase that has been implicated in growth and metastasis of various epithelial tumors. In this study, we have examined the role that FER kinase plays in melanoma at the molecular level. FER-depleted melanoma cells exhibit impaired Wnt/ß-catenin pathway activity, as well as multiple proteomic changes, which include decreased abundance of L1-cell adhesion molecule (L1-CAM). Consistent with the pro-metastatic functions of these pathways, we demonstrate that depletion of FER kinase decreases melanoma growth and formation of distant metastases in a xenograft model. These findings indicate that FER is an important positive regulator of melanoma metastasis and a potential target for innovative therapies.

Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/ß-catenin pathway, because ß-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

Pannexin 1 regulates adipose stromal cell differentiation and fat accumulation.

Pannexin 1 (Panx1) is a channel-forming glycoprotein important in paracrine signaling and cellular development. In this study, we discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain and blood markers of obesity as compared to WT controls, except for an increase in glucose and insulin levels. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high-fat diet. To uncover the cellular mechanism responsible for the increased fat content in the KO, we isolated primary cultures of adipose-derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. We concluded that Panx1 plays a key role in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.

Therapeutic response of metastatic angiomatoid fibrous histiocytoma carrying EWSR1-CREB1 fusion to the interleukin-6 receptor antibody tocilizumab.

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue tumor that has been associated with EWSR1-CREB1 gene fusion. Outcome in patients with unresectable distant metastases is generally fatal. Interleukin-6 (IL-6) secretion has been described in tumors with EWSR1-CREB1 fusion, and may promote tumor growth due to autocrine stimulation. Tocilizumab is an IL-6 receptor antibody that has potential benefit as a targeted therapy in refractory neoplasms with IL-6 secretion. We describe a child with metastatic AFH with EWSR1-CREB1 fusion and elevated IL-6 whose disease progressed during treatment with traditional chemotherapeutic agents, but improved after targeted therapy with tocilizumab.

N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1.

Pannexins (Panx1, 2, 3) are channel-forming glycoproteins expressed in mammalian tissues. We previously reported that N-glycosylation acts as a regulator of the localization and intermixing of Panx1 and Panx3, but its effects on Panx2 are currently unknown. Panx1 and Panx2 intermixing can regulate channel properties, and both pannexins have been implicated in neuronal cell death after ischemia. Our objectives were to validate the predicted N-glycosylation site of Panx2 and to study the effects of Panx2 glycosylation on localization and its capacity to interact with Panx1. We used site-directed mutagenesis, enzymatic de-glycosylation, cell-surface biotinylation, co-immunoprecipitation, and confocal microscopy. Our results showed that N86 is the only N-glycosylation site of Panx2. Panx2 and the N86Q mutant are predominantly localized to the endoplasmic reticulum (ER) and cis-Golgi matrix with limited cell surface localization was seen only in the presence of Panx1. The Panx2 N86Q mutant is glycosylation-deficient and tends to aggregate in the ER reducing its cell surface trafficking but it can still interact with Panx1. Our study indicates that N-glycosylation may be important for folding and trafficking of Panx2. We found that the un-glycosylated forms of Panx1 and 2 can readily interact, regulating their localization and potentially their channel function in cells where they are co-expressed.