Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using ß-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

Large-pore connexin hemichannels function like molecule transporters independent of ion conduction.

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore-a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.

Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels.

BACKGROUND: Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. RESULTS: Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. CONCLUSIONS: Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.

Thermal escape box: A cost-benefit evaluation paradigm for investigating thermosensation and thermal pain.

Thermosensation, the ability to detect and estimate temperature, is an evolutionarily conserved process that is essential for survival. Thermosensing is impaired in various pain syndromes, resulting in thermal allodynia, the perception of an innocuous temperature as painful, or thermal hyperalgesia, an exacerbated perception of a painful thermal stimulus. Several behavioral assays exist to study thermosensation and thermal pain in rodents, however, most rely on reflexive withdrawal responses or the subjective quantification of spontaneous nocifensive behaviors. Here, we created a new apparatus, the thermal escape box, which can be attached to temperature-controlled plates and used to assess temperature-dependent effort-based decision-making. The apparatus consists of a light chamber with an opening that fits around temperature-controlled plates, and a small entryway into a dark chamber. A mouse must choose to stay in a brightly lit aversive area or traverse the plates to escape to the enclosed dark chamber. We quantified escape latencies of adult C57Bl/6 mice at different plate temperatures from video recordings and found they were significantly longer at 5 °C, 18 °C, and 52 °C, compared to 30 °C, a mouse's preferred ambient temperature. Differences in escape latencies were abolished in male Trpm8-/- mice and in male Trpv1-/- animals. Finally, we show that chronic constriction injury procedures or oxaliplatin treatement significantly increased escape latencies at cold temperatures compared to controls, the later of which was prevented by the analgesic meloxicam. This demonstrates the utility of this assay in detecting cold pain. Collectively, our study has identified a new and effective tool that uses cost-benefit valuations to study thermosensation and thermal pain.

Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins secreted from dense granule organelles to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions, we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant "bubble vacuole" morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCE: Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels

Background Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthe-tizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. Results Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptordependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. Conclusions Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.

GRA47 and GRA72 are Toxoplasma gondii pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant 'bubble vacuole' morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections.

Remodeled connexin 43 hemichannels alter cardiac excitability and promote arrhythmias.

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the ß-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca2+ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.

A microtubule-connexin-43 regulatory link suppresses arrhythmias and cardiac fibrosis in Duchenne muscular dystrophy mice.

Dilated cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), an inherited degenerative disease of the cardiac and skeletal muscle caused by absence of the protein dystrophin. We showed one hallmark of DMD cardiomyopathy is the dysregulation of cardiac gap junction channel protein connexin-43 (Cx43). Proper Cx43 localization and function at the cardiac intercalated disc (ID) is regulated by post-translational phosphorylation of Cx43-carboxy-terminus residues S325/S328/S330 (pS-Cx43). Concurrently, Cx43 traffics along microtubules (MTs) for targeted delivery to the ID. In DMD hearts, absence of dystrophin results in a hyperdensified and disorganized MT cytoskeleton, yet the link with pS-Cx43 remains unaddressed. To gain insight into the relationship between MTs and pS-Cx43, DMD mice (mdx) and pS-Cx43-deficient (mdxS3A) mice were treated with an inhibitor of MT polymerization, colchicine (Colch). Colch treatment protected mdx, not mdxS3A mice, against Cx43 remodeling, improved MT directionality, and enhanced pS-Cx43/tubulin interaction. Likewise, severe arrhythmias were prevented in isoproterenol-stressed mdx, not mdxS3A mice. Furthermore, MT directionality was improved in pS-Cx43-mimicking mdx (mdxS3E). Mdxutr+/- and mdxutr+/-S3A mice, lacking one copy of dystrophin homolog utrophin, displayed enhanced cardiac fibrosis and reduced lifespan compared with mdxutr+/-S3E; and Colch treatment corrected cardiac fibrosis in mdxutr+/- but not mdxutr+/-S3A. Collectively, the data suggest that improved MT directionality reduces Cx43 remodeling and that pS-Cx43 is necessary and sufficient to regulate MT organization, which plays crucial role in correcting cardiac dysfunction in DMD mice. Thus, identification of novel organizational mechanisms acting on pS-Cx43-MT will help develop novel cardioprotective therapies for DMD cardiomyopathy.NEW & NOTEWORTHY We found that colchicine administration to Cx43-phospho-deficient dystrophic mice fails to protect against Cx43 remodeling. Conversely, Cx43-phospho-mimic dystrophic mice display a normalized MT network. We envision a bidirectional regulation whereby correction of the dystrophic MTs leads to correction of Cx43 remodeling, which in turn leads to further correction of the MTs. Our findings suggest a link between phospho-Cx43 and MTs that provides strong foundations for novel therapeutics in DMD cardiomyopathy.

Cx43 hemichannels contribute to astrocyte-mediated toxicity in sporadic and familial ALS.

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here, we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In addition, Cx43 expression is up-regulated in human postmortem tissue and cerebrospinal fluid from ALS patients. Using human induced pluripotent stem cell­derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we establish that Cx43 is up-regulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We also demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes using GAP 19, a mimetic peptide blocker, and tonabersat, a clinically tested small molecule, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Extending the in vitro application of tonabersat with chronic administration to SOD1G93A mice results in MN protection with a reduction in reactive astrocytosis and microgliosis. Taking these data together, our studies identify Cx43 hemichannels as conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

Mechanisms of ATP release in pain: role of pannexin and connexin channels.

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.

Free energy and kinetics of cAMP permeation through connexin26 via applied voltage and milestoning.

The connexin family is a diverse group of highly regulated wide-pore channels permeable to biological signaling molecules. Despite the critical roles of connexins in mediating selective molecular signaling in health and disease, the basis of molecular permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3',5'-cyclophosphate (cAMP), through a connexin26 hemichannel (Cx26). First, inward and outward fluxes of cAMP molecules solvated in KCl solution were obtained from 4 µs of ± 200 mV simulations. These fluxes data yielded a single-channel permeability of cAMP and cAMP/K+ permeability ratio consistent with experimentally measured values. The results from voltage simulations were then compared with the potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP without voltage, obtained from a total of 16.5 µs of Voronoi-tessellated Markovian milestoning simulations. Both the voltage simulations and the milestoning simulations revealed two cAMP-binding sites, for which the binding constants KD and dissociation rates koff were computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under opposite voltages were derived from the milestoning PMF and revealed the interplay between voltage and channel polarity on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and nonlocal pore diameter in a position-dependent manner.

Pannexin-1 Channels as Mediators of Neuroinflammation.

Neuroinflammation is a major component of central nervous system (CNS) injuries and neurological diseases, including Alzheimer's disease, multiple sclerosis, neuropathic pain, and brain trauma. The activation of innate immune cells at the damage site causes the release of pro-inflammatory cytokines and chemokines, which alter the functionality of nearby tissues and might mediate the recruitment of leukocytes to the injury site. If this process persists or is exacerbated, it prevents the adequate resolution of the inflammation, and ultimately enhances secondary damage. Adenosine 5' triphosphate (ATP) is among the molecules released that trigger an inflammatory response, and it serves as a chemotactic and endogenous danger signal. Extracellular ATP activates multiple purinergic receptors (P2X and P2Y) that have been shown to promote neuroinflammation in a variety of CNS diseases. Recent studies have shown that Pannexin-1 (Panx1) channels are the principal conduits of ATP release from dying cells and innate immune cells in the brain. Herein, we review the emerging evidence that directly implicates Panx-1 channels in the neuroinflammatory response in the CNS.

A method for assessing ionic and molecular permeation in connexin hemichannels.

Connexin hemichannels are permeable to both atomic ions and small molecules. Yet, they have different selectivity for ions and signaling molecules critical for biological functions. Activity of connexin hemichannels in living cells is commonly evaluated by methods that include electrophysiology and fluorescence-based approaches. Although less common, luminescence and radioactivity-based uptake/release assays have been also successfully used to determine selectivity and permeability to different molecules. The current methods, however, have important technical and quantitative limitations that make them unsuitable for simultaneously evaluating ionic and molecular permeability using different stimuli that control channel gating (e.g., voltage or extracellular Ca2+). To address this, we have recently designed a novel methodology that combines two-electrode voltage clamp (TEVC) and dye uptake assays in translucent Xenopus oocytes. This method allows for the evaluation of molecular transport kinetics in connexin hemichannels, and its utility can also be extended to other large pore channels, such as those formed by pannexin and CALHM. In this article, we describe step by step the protocol to perform the TEVC/Dye uptake assay.

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels.

Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease-causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma.

BACKGROUND: Neuroinflammation is a major component of secondary damage after traumatic brain injury (TBI). We recently reported that pharmacological inhibition of Pannexin-1 (Panx1) channels markedly reduced the inflammatory response after TBI. Panx1 channels have been shown to be important conduits for adenosine 5'-triphosphate (ATP) release and are associated with leukocyte infiltration and pyroptosis. Because Panx1 blockers significantly decrease ATP release and migration of activated microglia and other myeloid cells (such as monocyte-derived macrophages and dendritic cells) in vitro, we hypothesized that myeloid Panx1 channels play a specific role in immune cell infiltration promoting tissue damage following TBI. METHODS: The murine-controlled cortical impact (CCI) model was used on myeloid-specific Panx1 conditional knockout (Cx3cr1-Cre::Panx1fl/fl) mice to determine whether myeloid Panx1 mediates neuroinflammation and brain damage. Immune cell infiltration was measured using flow cytometry. Locomotor and memory functions were measured using the rotarod and Barnes maze test, respectively. The levels of biomarkers for tissue damage and blood-brain barrier leakage were measured using western blot and magnetic resonance imaging. Panx1 channel activity was measured with ex vivo dye uptake assays, using flow cytometry and confocal microscopy. RESULTS: CCI-injured Cx3cr1-Cre::Panx1fl/fl mice showed markedly reduced immune cell infiltration to the brain parenchyma compared with Panx1fl/fl mice. As expected, Panx1 dependent activity, assessed by dye uptake, was markedly reduced only in myeloid cells from Cx3cr1-Cre::Panx1fl/fl mice. The expression of biomarkers of tissue damage was significantly reduced in the CCI-injured Cx3cr1-Cre::Panx1fl/fl mice compared with Panx1fl/fl mice. In line with this, magnetic resonance imaging showed reduced blood-brain barrier leakage in CCI-injured Cx3cr1-Cre::Panx1fl/fl mice. There was also a significant improvement in motor and memory function in Cx3cr1-Cre::Panx1fl/fl mice when compared with Panx1fl/fl mice within a week post-CCI injury. CONCLUSION: Our data demonstrate that CCI-related outcomes correlate with Panx1 channel function in myeloid cells, indicating that activation of Panx1 channels in myeloid cells is a major contributor to acute brain inflammation following TBI. Importantly, our data indicate myeloid Panx1 channels could serve as an effective therapeutic target to improve outcome after TBI.