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Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

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Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.

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Connexins and pannexins are transmembrane proteins that can form direct (gap junctions) or indirect (connexons, pannexons) intercellular communication channels. By propagating ions, metabolites, sugars, nucleotides, miRNAs, and/or second messengers, they participate in a variety of physiological functions, such as tissue homeostasis and host defense. There is solid evidence supporting a role for intercellular signaling in various pulmonary inflammatory diseases where alteration of connexin/pannexin channel functional expression occurs, thus leading to abnormal intercellular communication pathways and contributing to pathophysiological aspects, such as innate immune defense and remodeling. The integrity of the airway epithelium, which is the first line of defense against invading microbes, is established and maintained by a repair mechanism that involves processes such as proliferation, migration, and differentiation. Here, we briefly summarize current knowledge on the contribution of connexins and pannexins to necessary processes of tissue repair and speculate on their possible involvement in the shaping of the airway epithelium integrity.

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Introduction: The subventricular zone (SVZ) is a brain region that contains neural stem cells and progenitor cells (NSCs/NPCs) from which new neurons and glial cells are formed during adulthood in mammals. Recent data indicate that SVZ NSCs are the cell type that acquires the initial tumorigenic mutation in glioblastoma (GBM), the most aggressive form of malignant glioma. NSCs/NPCs of the SVZ present hemichannel activity whose function has not yet been fully elucidated. In this work, we aimed to analyze whether hemichannel-mediated communication affects proliferation of SVZ NPCs and GBM cells. Methods and Results: For that purpose, we used boldine, an alkaloid derived from the boldo tree (Peumus boldus), that inhibits connexin and pannexin hemichannels, but without affecting gap junctional communication. Boldine treatment (50 µM) of rat SVZ NPCs grown as neurospheres effectively inhibited dye uptake through hemichannels and induced a significant reduction in neurosphere diameter and in bromodeoxyuridine (BrdU) incorporation. However, the differentiation pattern was not modified by the treatment. Experiments with specific blockers for hemichannels formed by connexin subunits (D4) or pannexin 1 (probenecid) revealed that probenecid, but not D4, produced a decrease in BrdU incorporation similar to that obtained with boldine. These results suggest that inhibition of pannexin 1 hemichannels could be partially responsible for the antiproliferative effect of boldine on SVZ NPCs. Analysis of the effect of boldine (25-600 µM) on different types of primary human GBM cells (GBM59, GBM96, and U87-MG) showed a concentration-dependent decrease in GBM cell growth. Boldine treatment also induced a significant inhibition of hemichannel activity in GBM cells. Discussion: Altogether, we provide evidence of an antimitotic action of boldine in SVZ NPCs and in GBM cells which may be due, at least in part, to its hemichannel blocking function. These results could be of relevance for future possible strategies in GBM aimed to suppress the proliferation of mutated NSCs or glioma stem cells that might remain in the brain after tumor resection.

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Ionotropic purinergic receptors (P2XRs) are activated by ATP and ATP analogs. ATP can be released through ATP-permeable channels such as the pannexin hemichannels. Upon activation, the P2XRs become permeable to Ca2+, a potent stimulator of mucin secretion in conjunctival goblet cells (CGCs). The purpose of this study was to investigate the presence and function of P2XRs in CGCs. We also examined the presence of pannexin hemichannels. Rat first passage CGCs were stained with the goblet cell marker anti-cytokeratin 7 antibody and specific antibodies to P2X1-7 receptors and pannexin 1-3. mRNA expression was determined by RT-PCR using primers specific to P2XRs and pannexins. Proteins were identified with Western blotting (WB) using the same antibodies as for immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with Fura 2-AM, exposed to agonists and antagonists, and intracellular [Ca2+] ([Ca2+]i) measured. [Ca2+]i was also measured after knock down of P2X4 and P2X7 receptor expression, and when exploiting P2XR specific characteristics. Lastly, mucin secretion was measured after the addition of several P2XR agonists. All P2XRs and pannexins were visualized with IF microscopy, and identified with RT-PCR and WB. [Ca2+]i was significantly increased when stimulated with ATP (10-7-10-4 M). Suramin, a non-selective P2XR antagonist at 10-4 M did not reduce ATP-induced peak [Ca2+]i. The potent P2X7 agonist, BzATP (10-7-10-4 M) increased the [Ca2+]i, although to a lesser extent than ATP. When measuring [Ca2+]i the effect of repeated applications of ATP at 10-5 or 10-6 M the response "desensitized" after 30-60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca2+]i increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca2+]i. ATP (10-5 M), BzATP (10-4 M) and 2MeSATP (10-5 M) induced mucin secretion. We conclude that all seven P2XRs are present in cultured rat CGCs. Of the P2XRs, only activation of the homotrimeric P2X4R appears to increase [Ca2+]i and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.

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Pannexins are an interesting new target in medicinal chemistry, as they are involved in many pathologies such as epilepsy, ischemic stroke, cancer and Parkinson's disease, as well as in neuropathic pain. They are a family of membrane channel proteins consisting of three members, Panx-1, Panx-2 and Panx-3, and are expressed in vertebrates. In the present study, as a continuation of our research in this field, we report the design, synthesis and pharmacological evaluation of new quinoline-based Panx-1 blockers. The most relevant compounds 6f and 6g show an IC50 = 3 and 1.5 µM, respectively, and are selective Panx-1 blockers. Finally, chemical stability, molecular modelling and X-ray crystallography studies have been performed providing useful information for the realization of the project.

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Hypertension is one of the most common risk factors for developing chronic cardiovascular diseases, including hypertensive nephropathy. Within the glomerulus, hypertension causes damage and activation of mesangial cells (MCs), eliciting the production of large amounts of vasoactive and proinflammatory agents. Accordingly, the activation of AT1 receptors by the vasoactive molecule angiotensin II (AngII) contributes to the pathogenesis of renal damage, which is mediated mostly by the dysfunction of intracellular Ca2+ ([Ca2+]i) signaling. Similarly, inflammation entails complex processes, where [Ca2+]i also play crucial roles. Deregulation of this second messenger increases cell damage and promotes fibrosis, reduces renal blood flow, and impairs the glomerular filtration barrier. In vertebrates, [Ca2+]i signaling depends, in part, on the activity of two families of large-pore channels: hemichannels and pannexons. Interestingly, the opening of these channels depends on [Ca2+]i signaling. In this review, we propose that the opening of channels formed by connexins and/or pannexins mediated by AngII induces the ATP release to the extracellular media, with the subsequent activation of purinergic receptors. This process could elicit Ca2+ overload and constitute a feed-forward mechanism, leading to kidney damage.

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The depletion of the endoplasmic reticulum (ER) Ca 2+ stores is known to activate a Ca 2+ route of the plasma membrane known as store-operated Ca 2+ entry (SOCE). Stromal interaction molecules (STIM1-2) and Orai1-3 proteins are regarded as the central molecular core components of SOCE. In a recent article, Patel and colleagues have identified a new type of coupling linking the Ca 2+ status of the ER and the activity of pannexin 1 (Panx1) ion channels distinct from Orai. This work further illustrates that Orai channels are far from being the exclusive partners of STIM proteins since these ER Ca 2+ sensors interact with a large diversity of targets and control several biological responses independently of Orai channels. Patel et al present an exciting new perspective on the contribution of the ER Ca 2+ release that recruits distinct types of cell surface ion channels such as Ca 2+ -selective (Orai) and nonselective (Panx1) channels. This study provides new insight into the complexity of store-operated ion channels signalling. Future studies will be required to better understand the contribution of this neuronal store-operated Panx1 response in neuronal pathophysiology.

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Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

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The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration ([Formula: see text]). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the [Formula: see text] in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain [Formula: see text] oscillations.

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Identifying signaling pathways and molecules involved in SARS-CoV-2 pathogenesis is pivotal for developing new effective therapeutic or preventive strategies for COVID-19. Pannexins (PANX) are ATP-release channels in the plasma membrane essential in many physiological and immune responses. Activation of pannexin channels and downstream purinergic receptors play dual roles in viral infection, either by facilitating viral replication and infection or inducing host antiviral defense. The current review provides a hypothesis demonstrating the possible contribution of the PANX1 channel and purinergic receptors in SARS-CoV-2 pathogenesis and mechanism of action. Moreover, we discuss whether targeting these signaling pathways may provide promising preventative therapies and treatments for patients with progressive COVID-19 resulting from excessive pro-inflammatory cytokines and chemokines production. Several inhibitors of this pathway have been developed for the treatment of other viral infections and pathological consequences. Specific PANX1 inhibitors could be potentially included as part of the COVID-19 treatment regimen if, in future, studies demonstrate the role of PANX1 in COVID-19 pathogenesis. Of note, any ATP therapeutic modulation for COVID-19 should be carefully designed and monitored because of the complex role of extracellular ATP in cellular physiology.

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Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.

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Pain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing.

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Adult skeletal muscle has robust regenerative capabilities due to the presence of a resident stem cell population called satellite cells. Muscle injury leads to these normally quiescent cells becoming molecularly and metabolically activated and embarking on a program of proliferation, migration, differentiation, and fusion culminating in the repair of damaged tissue. These processes are highly coordinated by paracrine signaling events that drive cytoskeletal rearrangement and cell-cell communication. Pannexins are a family of transmembrane channel proteins that mediate paracrine signaling by ATP release. It is known that Pannexin1 (Panx1) is expressed in skeletal muscle, however, the role of Panx1 during skeletal muscle development and regeneration remains poorly understood. Here we show that Panx1 is expressed on the surface of myoblasts and its expression is rapidly increased upon induction of differentiation and that Panx1-/- mice exhibit impaired muscle regeneration after injury. Panx1-/- myoblasts activate the myogenic differentiation program normally, but display marked deficits in migration and fusion. Mechanistically, we show that Panx1 activates P2 class purinergic receptors, which in turn mediate a lipid signaling cascade in myoblasts. This signaling induces bleb-driven amoeboid movement that in turn supports myoblast migration and fusion. Finally, we show that Panx1 is involved in the regulation of cell-matrix interaction through the induction of ADAMTS (Disintegrin-like and Metalloprotease domain with Thrombospondin-type 5) proteins that help remodel the extracellular matrix. These studies reveal a novel role for lipid-based signaling pathways activated by Panx1 in the coordination of myoblast activities essential for skeletal muscle regeneration.

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Maternal inflammation during pregnancy causes later-in-life alterations of the offspring's brain structure and function. These abnormalities increase the risk of developing several psychiatric and neurological disorders, including schizophrenia, intellectual disability, bipolar disorder, autism spectrum disorder, microcephaly, and cerebral palsy. Here, we discuss how astrocytes might contribute to postnatal brain dysfunction following maternal inflammation, focusing on the signaling mediated by two families of plasma membrane channels: hemi-channels and pannexons. [Ca2+]i imbalance linked to the opening of astrocytic hemichannels and pannexons could disturb essential functions that sustain astrocytic survival and astrocyte-to-neuron support, including energy and redox homeostasis, uptake of K+ and glutamate, and the delivery of neurotrophic factors and energy-rich metabolites. Both phenomena could make neurons more susceptible to the harmful effect of prenatal inflammation and the experience of a second immune challenge during adulthood. On the other hand, maternal inflammation could cause excitotoxicity by producing the release of high amounts of gliotransmitters via astrocytic hemichannels/pannexons, eliciting further neuronal damage. Understanding how hemichannels and pannexons participate in maternal inflammation-induced brain abnormalities could be critical for developing pharmacological therapies against neurological disorders observed in the offspring.

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Pannexins are a family of glycoproteins that comprises three members, PANX1, PANX2, and PANX3. The widely expressed and interrogated PANX1 forms heptameric membrane channels that primarily serve to connect the cytoplasm to the extracellular milieu by being selectively permeable to small signaling molecules when activated. Apart from notable exceptions, PANX1 in many tumor cells appears to facilitate tumor growth and metastasis, suggesting that pannexin-blocking therapeutics may have utility in cancer. Attenuation of PANX1 function must also consider the fact that PANX1 is found in stromal cells of the tumor microenvironment (TME), including immune cells. This review highlights the key discoveries of the past 5 years that suggest pannexins facilitate, or in some cases inhibit, tumor cell growth and metastasis via direct protein interactions and through the regulated efflux of signaling molecules.

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Human immunodeficiency virus-1 (HIV-1) has been shown to cross the blood-brain barrier and cause HIV-associated neurocognitive disorders (HAND) through a process that may involve direct or indirect interactions with the central nervous system (CNS) cells and alterations of amyloid ß (Aß) homeostasis. The present study focused on the mechanisms of HIV-1 infecting human neural progenitor cells (hNPCs) and affecting NPC intercellular communications with human brain endothelial cells (HBMEC). Despite the lack of the CD4 receptor, hNPCs were effectively infected by HIV-1 via a mechanism involving the chemokine receptors, CXCR4 and CCR5. HIV-1 infection increased expression of connexin-43 (Cx43), phosphorylated Cx43 (pCx43), and pannexin 2 (Panx2) protein levels in hNPCs, suggesting alterations in gap-junction (GJ) and pannexin channel communication. Indeed, a functional GJ assay indicated an increase in communication between HIV-infected hNPCs and non-infected HBMEC. We next analyzed the impact of HBMEC-derived extracellular vesicles (EVs) and EVs carrying Aß (EV-Aß) on the expression of Cx43, pCx43, and Panx2 in HIV-1 infected and non-infected hNPCs. Exposure to EV-Aß resulted in significant reduction of Cx43 and pCx43 protein expression in non-infected hNPCs when compared to EV controls. Interestingly, EV-Aß treatment significantly increased levels of Cx43, pCx43, and Panx2 in HIV-1-infected hNPCs when compared to non-infected controls. These results were confirmed in a GJ functional assay and an ATP release assay, which is an indicator of connexin hemichannel and/or pannexin channel functions. Overall, the current study demonstrates the importance of hNPCs in HIV-1 infection and indicates that intercellular communications between infected hNPCs and HBMEC can be effectively modulated by EVs carrying Aß as their cargo.

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In the nervous system, the concentration of Cl- in neurons that express GABA receptors plays a key role in establishing whether these neurons are excitatory, mostly during early development, or inhibitory. Thus, much attention has been dedicated to understanding how neurons regulate their intracellular Cl- concentration. However, regulation of the extracellular Cl- concentration by other cells of the nervous system, including glia and microglia, is as important because it ultimately affects the Cl- equilibrium potential across the neuronal plasma membrane. Moreover, Cl- ions are transported in and out of the cell, via either passive or active transporter systems, as counter ions for K+ whose concentration in the extracellular environment of the nervous system is tightly regulated because it directly affects neuronal excitability. In this book chapter, we report on the Cl- channel types expressed in the various types of glial cells focusing on the role they play in the function of the nervous system in health and disease. Furthermore, we describe the types of stimuli that these channels are activated by, the other solutes that they may transport, and the involvement of these channels in processes such as pH regulation and Regulatory Volume Decrease (RVD). The picture that emerges is one of the glial cells expressing a variety of Cl- channels, encoded by members of different gene families, involved both in short- and long-term regulation of the nervous system function. Finally, we report data on invertebrate model organisms, such as C. elegans and Drosophila, that are revealing important and previously unsuspected functions of some of these channels in the context of living and behaving animals.

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Connexins and Pannexins play important roles in osteocytes and osteoblasts differentiation, intracellular signal transduction, maintenance of bone balance, and bone regeneration. This article reviews the progress and limitations of Connexins-mediated gap junctions and Pannexins mediated hemichannel in bone. Current research has shown that these molecules, in the form of gap junctions or separate hemichannels, deliver external stimuli to the skeletal system. However, little is known about the role of other cell types in bone development and homeostasis, such as pre-osteoblasts and bone marrow mesenchymal stem cells, in maintaining normality. In addition, at present, the most well-studied member of the Connexins family is Connexin43 (Cx43), while the roles and mechanisms of other members in bone development are still behind the veil. Gene-edited animal models provide basic information on the role of Connexins and Pannexins in the skeletal system, but the similarities and differences between Connexins and Pannexins remain to be discovered. Targeting a specific function of Connexins or Pannexins for bone stimulation and bone disease remains a challenge, with pharmacological selective overlap between channels, compensation of other subtypes, differences in methods for assessing channel function, and genetic changes associated with transgenic mouse models. Therefore, better tools and research pathways are needed to understand the role of these pathways in bone and cartilage. An essential task for future research will be to identify specific compounds that regulate Connexins or Pannexins subtypes to enable them to be used as pharmaceutical agents in the treatment of bone diseases, providing the possibility to develop new therapeutic strategies for improving bone health and treating diseases of the skeletal system.

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The integration of neurons into networks relies on the formation of dendritic spines. These specialized structures arise from dynamic filopodia-like dendritic protrusions. It was recently reported that cortical neurons lacking the channel protein pannexin 1 (PANX1) exhibited higher dendritic spine densities. Here, we expanded on those findings to investigate, at an earlier developmental time point (with more abundant dendritic protrusions), whether differences in the properties of dendritic protrusion dynamics could contribute to this previously discovered phenomenon. Using a fluorescent membrane tag (mCherry-CD9-10) to visualize dendritic protrusions in developing neurons [at 10 d in vitro (DIV10)], we confirmed that lack of PANX1 led to higher protrusion density, while transient transfection of Panx1 led to decreased protrusion density. To quantify the impact of PANX1 expression on protrusion formation, elimination, and motility, we used live cell imaging in DIV10 neurons (one frame every 5 s for 10 min). We discovered that at DIV10, loss of PANX1 stabilized protrusions. Notably, re-expression of PANX1 in Panx1 knock-out (KO) neurons resulted in a significant increase in protrusion motility and turnover. In summary, these new data revealed that PANX1 could regulate the development of dendritic spines, in part, by controlling dendritic protrusion dynamics.

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