Panx1 Gene Research Articles

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.

Pannexin 1 Transgenic Mice: Human Diseases and Sleep-Wake Function Revision.

In humans and other vertebrates pannexin protein family was discovered by homology to invertebrate gap junction proteins. Several biological functions were attributed to three vertebrate pannexins members. Six clinically significant independent variants of the PANX1 gene lead to human infertility and oocyte development defects, and the Arg217His variant was associated with pronounced symptoms of primary ovarian failure, severe intellectual disability, sensorineural hearing loss, and kyphosis. At the same time, only mild phenotypes were observed in Panx1 knockout mice. In addition, a passenger mutation was identified in a popular line of Panx1 knockout mice, questioning even those effects. Using CRISPR/Cas9, we created a new line of Panx1 knockout mice and a new line of mice with the clinically significant Panx1 substitution (Arg217His). In both cases, we observed no significant changes in mouse size, weight, or fertility. In addition, we attempted to reproduce a previous study on sleep/wake and locomotor activity functions in Panx1 knockout mice and found that previously reported effects were probably not caused by the Panx1 knockout itself. We consider that the pathological role of Arg217His substitution in Panx1, and some Panx1 functions in general calls for a re-evaluation.

Pannexin 1 Influences Lineage Specification of Human iPSCs.

Every single cell in the body communicates with nearby cells to locally organize activities with their neighbors and dysfunctional cell-cell communication can be detrimental during cell lineage commitment, tissue patterning and organ development. Pannexin channels (PANX1, PANX2, and PANX3) facilitate purinergic paracrine signaling through the passage of messenger molecules out of cells. PANX1 is widely expressed throughout the body and has recently been identified in human oocytes as well as 2 and 4-cell stage human embryos. Given its abundance across multiple adult tissues and its expression at the earliest stages of human development, we sought to understand whether PANX1 impacts human induced pluripotent stem cells (iPSCs) or plays a role in cell fate decisions. Western blot, immunofluorescence and flow cytometry reveal that PANX1 is expressed in iPSCs as well as all three germ lineages derived from these cells: ectoderm, endoderm, and mesoderm. PANX1 demonstrates differential glycosylation patterns and subcellular localization across the germ lineages. Using CRISPR-Cas9 gene ablation, we find that loss of PANX1 has no obvious impact on iPSC morphology, survival, or pluripotency gene expression. However, PANX1 gene knockout iPSCs exhibit apparent lineage specification bias under 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 increases representation of endodermal and mesodermal populations in PANX1 knockout cells. Importantly, PANX1 knockout iPSCs are fully capable of differentiating toward each specific lineage when exposed to the appropriate external signaling pressures, suggesting that although PANX1 influences germ lineage specification, it is not essential to this process.

Activity–Rest and Body Temperature Rhythms in Mice with Knockout of the Panx1 Gene

Pannexins are a family of three proteins which play an important role in intercellular communications. This study shows that mice with knockout of the Panx1 gene(Panx1–/–) have elevated levels of movement activity and increased proportions of waking, due to decreases in slow-wave sleep, as compared with control animals, as a result of decreases in the adenosine content of the brain. We suggest that these mice may also show impairments to thermoregulation and the daily temperature curve rhythm. To test this suggestion, eight adult male C57BL/6J mice and eight adult male Panx1–/– mice, derived from them, underwent i.p. implantation of autonomous temperature and movement activity sensors. Recordings were made over several months at an environmental temperature of 23 ± 1°C. The results confirmed our previous data indicating higher movement activity in Panx1–/– mice compared with controls. The daily body temperature rhythm in Panx1–/– mice persisted and was no different from that in control animals. The first series of experiments showed an increased tendency to fall into torpor in Panx1–/– mice as compared with controls. However, no onset of torpor occurred in the next series, using a further four pairs of animals. Thus, the role of the Panx1 gene in the diurnal activity-rest rhythm was confirmed, while its role in the temperature rhythm and thermoregulatory reactions remains unclear and requires further study.

Distinct roles for innexin gap junctions and hemichannels in mechanosensation.

Mechanosensation is central to a wide range of functions, including tactile and pain perception, hearing, proprioception, and control of blood pressure, but identifying the molecules underlying mechanotransduction has proved challenging. In Caenorhabditis elegans, the avoidance response to gentle body touch is mediated by six touch receptor neurons (TRNs), and is dependent on MEC-4, a DEG/ENaC channel. We show that hemichannels containing the innexin protein UNC-7 are also essential for gentle touch in the TRNs, as well as harsh touch in both the TRNs and the PVD nociceptors. UNC-7 and MEC-4 do not colocalize, suggesting that their roles in mechanosensory transduction are independent. Heterologous expression of unc-7 in touch-insensitive chemosensory neurons confers ectopic touch sensitivity, indicating a specific role for UNC-7 hemichannels in mechanosensation. The unc-7 touch defect can be rescued by the homologous mouse gene Panx1 gene, thus, innexin/pannexin proteins may play broadly conserved roles in neuronal mechanotransduction.

The Sleep–Waking Cycle and Experimental Models of Mutations in the Panx1 Gene

Objectives. To confirm the authors’ previous suggestion that mutations in the Panx1 gene (pannexin-1) may play an important role in the development of impairments to the sleep-waking cycle. Materials and methods. Studies were carried out using mice with knockout of the Panx1 gene. Experiments used continuous 24-h EEG recording of the EEG and motor activity. Results and conclusions. A statistically significant increase in the proportion of waking was found, due to a decrease in the slow-wave sleep phase (as compared with controls), particularly marked in the dark period of the day, along with an increase in motor activity. The results were compared with data obtained during investigation of a recently published case of a patient with a homozygous mutation in this gene. The possible physiological role of pannexin-1 protein in health and pathology is discussed.

Expression analysis of Pannexin1 channel gene in response to immune challenges and its role in infection-induced ATP release in tilapia (Oreochromis niloticus)

ATP released from immune cells plays an important role in activation of host innate immunity. However, the molecular mechanisms for pathogen infection-induced ATP release in fish remains unclear. Pannexin1 (Panx1) is a recently identified ATP release channel important for controlling immune responses. The immune relevance of Panx1 in fish, however, is still poorly understood. In this study, we characterized a Panx1 gene homologue (termed tPanx1) from Nile tilapia (Oreochromis niloticus) and analyzed its expression in response to different immune challenges. We also investigated the role of tPanx1 channel in bacterial infection-induced ATP release. Real-time quantitative PCR analysis revealed that tPanx1 gene is expressed in all tested tissues with predominant expression in intestine. Immune challenges with lipopolysaccharide, polyinosinic-polycytidylic acid and zymosan led to increased gene expression of tPanx1 in tilapia head kidney cells and peripheral blood leucocytes. In addition, tPanx1 gene was up-regulated in hepatopancreas, muscle, spleen, gill, head kidney and blood after Aeromonas hydrophila infection. Furthermore, pharmacological inhibition of tPanx1 channel activity with Panx1 channel inhibitor, carbenoxolone, significantly attenuated A. hydrophila infection-induced ATP release in tilapia head kidney cells. Taken together, our findings suggested that tPanx1 is an important immune response gene involved in bacterial infection-induced ATP release in tilapia O. niloticus.

Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.

Pannexin 1 sustains the electrophysiological responsiveness of retinal ganglion cells

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

РИТМЫ АКТИВНОСТИ-ПОКОЯ И ТЕМПЕРАТУРЫ ТЕЛА У МЫШЕЙ, НОКАУТНЫХ ПО ГЕНУ PANXI

Pannexins are a protein family playing an important role in the intercellular communication. We have previously demonstrated the elevated locomotor activity and wake time percentage at the expense of NREM sleep in Panx1­/­ as compared to WT mice. This effect is possibly due to the decreased level of cerebral adenosine in Panx1­/­ mice. Thus we also proposed possible disturbances in thermoregulation and the circadian temperature curve. To check up the hypothesis, 8 adult male Panx1­/­ and 8 correspondent WT mice; were implanted with intraperitoneal autonomic temperature and movement probes under anesthesia. The recording were performed for several months at the room temperature (23 ) 1°С). The results confirmed our previous data on higher movement activity in Panx1­/­ vs. WT mice. Circadian rhythm of body temperature was the same in both groups of mice. Increased tendency to fall into torpor in Panx1­/­ vs. WT mice was observed in the four pairs of animals that were studied after the summer solstice. Howevwe, no falls were observed in another four pairs of mice studied after the winter solstice. Thus, the role of Panx1 gene in the circadian rhythm of rest-activity was confirmed, although its role in the temperature rhythm and thermoregulatory reactions remains unclear and requires further investigation.

Pannexin 1 Modulates Axonal Growth in Mouse Peripheral Nerves.

The pannexin family of channels consists of three members—pannexin-1 (Panx1), pannexin-2 (Panx2), and pannexin-3 (Panx3) that enable the exchange of metabolites and signaling molecules between intracellular and extracellular compartments. Pannexin-mediated release of intracellular ATP into the extracellular space has been tied to a number of cellular activities, primarily through the activity of type P2 purinergic receptors. Previous work indicates that the opening of Panx1 channels and activation of purinergic receptors by extracellular ATP may cause inflammation and apoptosis. In the CNS (central nervous system) and PNS (peripheral nervous system), coupled pannexin, and P2 functions have been linked to peripheral sensitization (pain) pathways. Purinergic pathways are also essential for other critical processes in the PNS, including myelination and neurite outgrowth. However, whether such pathways are pannexin-dependent remains to be determined. In this study, we use a Panx1 knockout mouse model and pharmacological inhibitors of the Panx1 and the ATP-mediated signaling pathway to fill gaps in our understanding of Panx1 localization in peripheral nerves, roles for Panx1 in axonal outgrowth and myelination, and neurite extension. Our data show that Panx1 is localized to axonal, myelin, and vascular compartments of the peripheral nerves. Knockout of Panx1 gene significantly increased axonal caliber in vivo and axonal growth rate in cultured dorsal root ganglia (DRG) neurons. Furthermore, genetic knockout of Panx1 or inhibition of components of purinergic signaling, by treatment with probenecid and apyrase, resulted in denser axonal outgrowth from cultured DRG explants compared to untreated wild-types. Our findings suggest that Panx1 regulates axonal growth in the peripheral nervous system.

Pannexin1 Single Nucleotide Polymorphism and Platelet Reactivity in a Cohort of Cardiovascular Patients.

Pannexin1 (Panx1), a membrane channel-forming protein permitting the passage of small-sized molecules, such as ATP, is expressed in human platelets. Recently, we showed that inhibiting Panx1 affects collagen-induced platelet aggregation but not aggregation triggered by other agonists. We also found that a single nucleotide polymorphism (SNP; rs1138800) in the Panx1 gene encoded for a gain-of-function channel (Panx1-400C) and was associated with enhanced collagen-induced platelet reactivity. Here, we assessed the association of this SNP with platelet reactivity in a cohort of 758 stable cardiovascular patients from the ADRIE study treated with aspirin and/or clopidogrel. We found that presence of the Panx1-400C allele was not associated with platelet reactivity in stable cardiovascular patients, irrespective of the platelet aggregation agonist used (collagen, ADP or arachidonic acid) or the anti-platelet drug regimen. Moreover, the Panx1-400A > C SNP did also not affect the re-occurrence of cardiac ischemic events in the same stable cardiovascular patient cohort.

Parental vitamin D deficiency during pregnancy is associated with increased blood pressure in offspring via Panx1 hypermethylation.

Vitamin D deficiency is one of the most common nutritional deficiencies worldwide. Maternal vitamin D deficiency is associated with increased susceptibility to hypertension in offspring, but the reasons for this remain unknown. The aim of this study was to determine if parental vitamin D deficiency leads to altered DNA methylation in offspring that may relate to hypertension. Male and female Sprague-Dawley rats were fed a standard or vitamin D-depleted diet. After 10 wk, nonsibling rats were mated. The conceived pups received standard chow. We observed an increased systolic and diastolic blood pressure in the offspring from depleted parents (F1-depl). Genome-wide methylation analyses in offspring identified hypermethylation of the promoter region of the Pannexin-1 (Panx1) gene in F1-depl rats. Panx1 encodes a hemichannel known to be involved in endothelial-dependent relaxation, and we demonstrated that in F1-depl rats the increase in blood pressure was associated with impaired endothelial relaxation of the large vessels, suggesting an underlying biological mechanism of increased blood pressure in children from parents with vitamin deficiency. Parental vitamin D deficiency is associated with epigenetic changes and increased blood pressure levels in offspring.

Functional role of a polymorphism in the Pannexin1 gene in collagen-induced platelet aggregation.

Pannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific (10)Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1-400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1-400A allele. Conversely, the frequency of Panx1-400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1-400C channels compared with Panx1-400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1-400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1-400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

The role of pannexin hemichannels in inflammation and regeneration

Tissue injury involves coordinated systemic responses including inflammatory response, targeted cell migration, cell-cell communication, stem cell activation and proliferation, and tissue inflammation and regeneration. The inflammatory response is an important prerequisite for regeneration. Multiple studies suggest that extensive cell-cell communication during tissue regeneration is coordinated by purinergic signaling via extracellular adenosine triphosphate (ATP). Most recent data indicates that ATP release for such communication is mediated by hemichannels formed by connexins and pannexins. The Pannexin family consists of three vertebrate proteins (Panx 1, 2, and 3) that have low sequence homology with other gap junction proteins and were shown to form predominantly non-junctional plasma membrane hemichannels. Pannexin-1 (Panx1) channels function as an integral component of the P2X/P2Y purinergic signaling pathway and is arguably the major contributor to pathophysiological ATP release. Panx1 is expressed in many tissues, with highest levels detected in developing brain, retina and skeletal muscles. Panx1 channel expression and activity is reported to increase significantly following injury/inflammation and during regeneration and differentiation. Recent studies also report that pharmacological blockade of the Panx1 channel or genetic ablation of the Panx1 gene cause significant disruption of progenitor cell migration, proliferation, and tissue regeneration. These findings suggest that pannexins play important roles in activation of both post-injury inflammatory response and the subsequent process of tissue regeneration. Due to wide expression in multiple tissues and involvement in diverse signaling pathways, pannexins and connexins are currently being considered as therapeutic targets for traumatic brain or spinal cord injuries, ischemic stroke and cancer. The precise role of pannexins and connexins in the balance between tissue inflammation and regeneration needs to be further understood.

Promises and pitfalls of a Pannexin1 transgenic mouse line

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1tm1a(KOMP)Wtsi. Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1f/f mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.

Function without form: an ongoing search for maxi-anion channel proteins. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

<i>Function without form: an ongoing search for maxi-anion channel proteins</i>. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

Pannexin 1 Ohnologs in the Teleost Lineage

Advances in genomic analysis indicate that the early chordate lineage underwent two whole-genome duplication events in fairly rapid succession around 400-600 million years ago, and that a third duplication event punctuated the radiation of ray-finned fishes (teleosts) around 320-350 million years ago. Connexin ohnologs have been disproportionately well maintained in the teleost genome following this third event, implying that gap junction proteins are amenable to neofunctionalization. A second family of gap junction-like proteins, the pannexins, is also present in chordates, but expansion of this family following the teleost whole-genome duplication has not been addressed in the literature. In the current study we report that ohnologs of panx1 are expressed by zebrafish, and orthologs of these two genes can be found in various other teleost species. The genomic locality of each gene is described, along with sequence alignments that reveal conservation of classic pannexin-specific features/motifs. The transcripts were then cloned from cDNA for in vitro analysis, and both are shown to traffic to the plasma membrane when exogenously expressed. Furthermore, electrophysiological recordings show differences in the biophysical properties between the channels formed by these two proteins. Our results indicate that both copies of the ancestral teleost panx1 gene were conserved following the last whole-genome duplication event and, following conventional zebrafish nomenclature, should now be referred to as panx1a and panx1b.

Complex interplay between Pannexin 1 cysteine residues and channel regulation

Post‐translational modification of proteins by S‐nitrosylation at unique cysteine residues can profoundly affect protein function. We recently demonstrated the presence of pannexin 1 (Panx1) channels in the vasculature and thus investigated whether Panx1 can be S‐nitrosylated and if this modification affects channel activity. Application of S‐nitrosogluathione (GSNO), a nitric oxide (NO) donor, to HEK cells transfected with wild type (WT) Panx1 caused S‐nitrosylation of the protein as detected by the biotin switch assay. To identify which cysteine was being S‐nitrosylated, we performed multiple cysteine to alanine mutations (C40A, C66A, C346A, C426A) in the murine Panx1 gene. Mutation of cysteine 426 blocked S‐nitrosylation of Panx1, indicating this residue as the site of modification. To assess functional effects of Panx1 S‐nitrosylation, we subjected HEK cells transfected with WT or our Panx1 cysteine mutants to whole‐cell patch clamp recordings. Application of GSNO dramatically attenuated the Panx1 current in HEK cells expressing WT Panx1. However, C426A current was still inhibited similar to WT Panx1 current, whereas C40A and C346A Panx1 channel current were not altered by GSNO. Lastly, we measured dye uptake and ATP release to correlate electrophysiology data. This data suggests a complex interplay between cysteine regulation of pore function and NO.

Dispensable ATP permeability of Pannexin 1 channels in a heterologous system and in mammalian taste cells

Afferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly. This inconsistency could indicate that the cellular context specifies Panx1 functioning. We cloned Panx1 from murine taste tissue, and heterologously expressed it in three different cell lines: HEK-293, CHO and neuroblastoma SK-N-SH cells. In all three cell lines, Panx1 transfection yielded outwardly rectifying anion channels that exhibited fast gating and negligible permeability to anions exceeding 250 Da. Despite expression of Panx1, the host cells did not liberate ATP upon stimulation, making it unclear whether Panx1 is involved in taste-related ATP secretion. This issue was addressed using mice with genetic ablation of the Panx1 gene. The ATP-biosensor assay revealed that, in taste cells devoid of Panx1, ATP secretion was robust and apparently unchanged compared with the control. Our data suggest that Panx1 alone forms a channel that has insufficient permeability to ATP. Perhaps, a distinct subunit and/or a regulatory circuit that is absent in taste cells is required to enable a high ATP-permeability mode of a native Panx1-based channel.