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Pannexin-1 (Panx1) hemichannel is a non-selective transmembrane channel that may play important roles in intercellular signaling by allowing the permeation of ions and metabolites, such as ATP. Although recent evidence shows that the Panx1 hemichannel is involved in controlling excitatory synaptic transmission, the role of Panx1 in inhibitory transmission remains unknown. Here, we studied the contribution of Panx1 to the GABAergic synaptic efficacy onto CA1 pyramidal neurons (PyNs) by using patch-clamp recordings and pharmacological approaches in wild-type and Panx1 knock-out (Panx1-KO) mice. We reported that blockage of the Panx1 hemichannel with the mimetic peptide 10 Panx1 increases the synaptic level of endocannabinoids (eCB) and the activation of cannabinoid receptors type 1 (CB1Rs), which results in a decrease in hippocampal GABAergic efficacy, shifting excitation/inhibition (E/I) balance toward excitation and facilitating the induction of long-term potentiation. Our finding provides important insight unveiling that Panx1 can strongly influence the overall neuronal excitability and play a key role in shaping synaptic changes affecting the amplitude and direction of plasticity, as well as learning and memory processes.

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca 2+ /calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca 2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved., Competing Interests: The authors declare no competing interest.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca 2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca 2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca 2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca 2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca 2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion., (© 2020 International Society for Neurochemistry.)

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Mast cells (MCs) release pro-inflammatory mediators through a process called degranulation response. The latter may be induced by several conditions, including antigen recognition through immunoglobulin E (IgE) or "cross-linking," classically associated with Type I hypersensitivity reactions. Early in this reaction, Ca 2+ influx and subsequent increase of intracellular free Ca 2+ concentration are essential for MC degranulation. Several membrane channels that mediate Ca 2+ influx have been proposed, but their role remains elusive. Here, we evaluated the possible contribution of pannexin-1 channels (Panx1 Chs), well-known as ATP-releasing channels, in the increase of intracellular Ca 2+ triggered during cross-linking reaction of MCs. The contribution of Panx1 Chs in the degranulation response was evaluated in MCs from wild type (WT) and Panx1 knock out (Panx1 -/- ) mice after anti-ovalbumin (OVA) IgE sensitization. Notably, the degranulation response (toluidine blue and histamine release) was absent in Panx1 -/- MCs. Moreover, WT MCs showed a rapid and transient increase in Ca 2+ signal followed by a sustained increase after antigen stimulation. However, the sustained increase in Ca 2+ signal triggered by OVA was absent in Panx1 -/- MCs. Furthermore, OVA stimulation increased the membrane permeability assessed by dye uptake, a prevented response by Panx1 Ch but not by connexin hemichannel blockers and without effect on Panx1 -/- MCs. Interestingly, the increase in membrane permeability of WT MCs was also prevented by suramin, a P2 purinergic inhibitor, suggesting that Panx1 Chs act as ATP-releasing channels impermeable to Ca 2+ . Accordingly, stimulation with exogenous ATP restored the degranulation response and sustained increase in Ca 2+ signal of OVA stimulated Panx1 -/- MCs. Moreover, opening of Panx1 Chs in Panx1 transfected HeLa cells increased dye uptake and ATP release but did not promote Ca 2+ influx, confirming that Panx1 Chs permeable to ATP are not permeable to Ca 2+ . These data strongly suggest that during antigen recognition, Panx1 Chs contribute to the sustained Ca 2+ signal increase via release of ATP that activates P2 receptors, playing a critical role in the sequential events that leads to degranulation response during Type I hypersensitivity reactions., (Copyright © 2019 Harcha, López, Sáez, Fernández, Barría, Martínez and Sáez.)