A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model

Whyte-Fagundes, Paige; Kurtenbach, Stefan; Zoidl, Christiane; Shestopalov, Valery I.; Carlen, Peter L.; Zoidl, Georg

doi:10.3389/fnmol.2018.00135

Cited by 22 publications

(18 citation statements)

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“…Neuro 2a cells transfected with fluorescently tagged WT panx1a and panx1a mutants were tested for dye uptake 48 h post-transfection using a protocol described previously [14,22]. Cells were grown on 35mm glass-bottom dishes and were incubated in clear DMEM lacking phenol red for 30min prior to imaging.…”

Section: Dye Uptake Assaymentioning

confidence: 99%

Role of an Aromatic–Aromatic Interaction in the Assembly and Trafficking of the Zebrafish Panx1a Membrane Channel

2020

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Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein. Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking.Biomolecules 2020, 10, 272 2 of 15 teleost whole-genome duplication event that occurred between 320 and 350 million years ago, a panx1a ohnologue exists (panx1b) [16]. Panx1a and panx1b show distinct tissue expressions, glycosylation patterns, and electrophysiological gating properties [14]. This study will focus on the panx1a ohnologue.Electrophysiological gating properties and multiple factors regulating the Panx1 channel, as well as complex pharmacology, have been described [17,18]. Panx1 blockers include carbenoxolone, mefloquine, and flufenamic acid, which also act on gap junction proteins. Potassium and glutamate can activate pannexins. Studies across multiple fields demonstrated that Panx1 is a major molecular hub interacting with many signaling pathways. To address the complex life cycle of Panx1, we explored the role of an aromatic-aromatic interaction between amino acids W123 and Y205 in the cytoplasmic loop of panx1a near transmembrane (TM) domains 2 and 3, respectively. Aromatic-aromatic interactions have been previously shown to be important in TM-TM association of membrane proteins [19,20]. The forces of these interactions help strengthen oligomerization and suggest a role in folding and stabilization. Both W123 and Y205 are highly conserved between various membrane channels and gap junction proteins. Mutation analysis of panx1a paired with co-localization and protein interaction studies led to the conclusion that the two aromatic residues are vital for the structural stabilization and interaction of panx1a TM domains before insertion into the cell membrane. Outcomes of this study are relevant to understand how Panx1 proteins mature and traffic to the cell membrane.

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Section: Dye Uptake Assaymentioning

confidence: 99%

Role of an Aromatic–Aromatic Interaction in the Assembly and Trafficking of the Zebrafish Panx1a Membrane Channel

2020

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“…Panx1 and Panx3 share protein sequence homology, and thus, it is postulated that the lack of gross overt phenotypes observed in one Panx null mouse modelcould be due to functional compensation. In fact, Panx3 was upregulated in the skin, blood vessels, and the vomeronasal organ of Panx1 −/− mice [17,30,31]. To address this compensation issue, we have generated the first Panx1 and Panx3 double knock-out mouse (dKO) and focused our characterization on the skin, bone, and the auditory system that are known to express both pannexins.…”

Section: Introductionmentioning

confidence: 99%

Double deletion of Panx1 and Panx3 affects skin and bone but not hearing

et al. 2019

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Pannexins (Panxs), large-pore channel forming glycoproteins, are expressed in a wide variety of tissues including the skin, bone, and cochlea. To date, the use of single knockout mouse models of both Panx1 and Panx3 have demonstrated their roles in skin development, bone formation, and auditory phenotypes. Due to sequence homology between Panx1 and Panx3, when one Panx is ablated from germline, the other may be upregulated in a compensatory mechanism to maintain tissue homeostasis and function. To evaluate the roles of Panx1 and Panx3 in the skin, bone, and cochlea, we created the first Panx1/Panx3 double knockout mouse model (dKO). These mice had smaller litters and reduced body weight compared to wildtype controls. The dKO dorsal skin had decreased epidermal and dermal area as well as decreased hypodermal area in neonatal but not in older mice. In addition, mouse skull shape and size were altered, and long bone length was decreased in neonatal dKO mice. Finally, auditory tests revealed that dKO mice did not exhibit hearing loss and were even slightly protected against noise-induced hearing damage at mid-frequency regions. Taken together, our findings suggest that Panx1 and Panx3 are important at early stages of development in the skin and bone but may be redundant in the auditory system. Key messages & Panx double KO mice had smaller litters and reduced body weight. & dKO skin had decreased epidermal and dermal area in neonatal mice. & Skull shape and size changed plus long bone length decreased in neonatal dKO mice. & dKO had no hearing loss and were slightly protected against noise-induced damage.

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“…2b). Compensatory regulation of three other pannexin genes similar to previous reports using Panx1 −/− mouse models was excluded 25 . Panx1b mRNA was expressed at equal levels in both wild-type and panx1a −/− .…”

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Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

Safarian

Whyte-Fagundes

Zoidl

et al. 2020

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Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology. Here, RNA-seq analysis of 6 days post fertilization larvae were confirmed by Real-Time PCR and paired with testing visual-motor behavior and in vivo electrophysiology. Results demonstrated that loss of panx1a specifically affected the expression of gene classes representing the development of the visual system and visual processing. Abnormal swimming behavior in the dark and the expression regulation of pre-and postsynaptic biomarkers suggested changes in dopaminergic signaling. Indeed, altered visuomotor behavior in the absence of functional Panx1a was evoked through D1/D2-like receptor agonist treatment and rescued with the D2-like receptor antagonist Haloperidol. Local field potentials recorded from superficial areas of the optic tectum receiving input from the retina confirmed abnormal responses to visual stimuli, which resembled treatments with a dopamine receptor agonist or pharmacological blocking of Panx1a. We conclude that Panx1a functions are relevant at a time point when neuronal networks supporting visualmotor functions undergo modifications preparing for complex behaviors of freely swimming fish. Pannexin 1 (Panx1) is an integral membrane glycoprotein forming ATP release channels in different tissues and cell types 1-5 , including neurons 6-8. In the CNS, evidence for physiological functions of Panx1 points at roles in the processing of sensory signals and learning and memory 9-11. For example, in Panx1 knockout mice, altered retinal contrast sensitivity 12 and hearing loss have been found 13,14. Also, performance in spatial learning and memory abilities such as object recognition and fear conditioning tasks are decreased 9,15,16. Intellectual disabilities, severe hearing loss, primary ovarian failure, kyphoscoliosis, and difficulties navigating in darkness were found in the first human patient identified with a homozygous Panx1 mutation 17. To form a better view of Panx1 functions in the processing of sensory information, we used the zebrafish as model organisms. Two Panx1 genes, panx1a and panx1b, originated from partial genome duplications during early teleost evolution 18,19. Although the two genes have been separated for more than 200 million years, principal channel functions are comparable to rodent or human Panx1 20. In the retina, the panx1a protein is expressed in horizontal cells 19,20 and plays essential roles in feedback from horizontal cells to cones in adult zebrafish 21-23. Here the panx1a gene was edited using transcription activator-like effector nucleases (TALEN). A loss of function mutation allowed to investigate Panx1a in 6 dpf old zebrafish larvae at a developmental stage when neuronal networks for visually guided locomotor behaviors were functional...

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A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model

Cited by 22 publications

References 68 publications

Role of an Aromatic–Aromatic Interaction in the Assembly and Trafficking of the Zebrafish Panx1a Membrane Channel

Role of an Aromatic–Aromatic Interaction in the Assembly and Trafficking of the Zebrafish Panx1a Membrane Channel

Double deletion of Panx1 and Panx3 affects skin and bone but not hearing

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

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