A dual voltage clamp technique to study gap junction hemichannels in astrocytes cultured from neonatal rodent spinal cords

Mapping Intimacies ◽

10.1101/2021.11.20.469295 ◽

2021 ◽

Author(s):

Juan Mauricio Garre ◽

Feliksas F Bukauskas ◽

Michael V Bennett

Keyword(s):

Voltage Clamp ◽

Connexin 43 ◽

Single Channel ◽

Purinergic Signaling ◽

Atp Release ◽

Single Channels ◽

P2x7 Receptors ◽

Voltage Clamp Technique ◽

Pannexin 1 ◽

Clamp Technique

Astrocytes express surface channels involved in purinergic signaling, and among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) mediate ATP release that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, i.e., open and close, under physiological and pathological conditions, single channel conductance of Px1 HCs is not well defined. Here, we developed a dual voltage clamp technique in HeLa cells overexpressing human Px1-YFP, and then applied this system to rodent spinal astrocytes. Single channels were recorded in cell attached patches and evoked with ramp cycles of 2 s duration and -/+ 80-100 mV amplitude or rectangular pulses through another pipette in whole cell clamp. Conductance of Px1 HC openings recorded during ramp stimuli ranged 25-110 pS. Based on their single channel conductances, Px1 HCs could be distinguished from Cx43 HCs and P2X7 receptors (P2X7Rs) in spinal astrocytes during dual voltage clamp experiments. Furthermore, we found that single channel activity of Cx43 HCs and P2X7Rs was increased, and that of Px1 HCs was decreased, in spinal astrocytes treated for 7h with FGF-1, a growth factor implicated in neurodevelopment, repair and inflammation.

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HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function

International Journal of Molecular Sciences ◽

10.3390/ijms21072503 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2503 ◽

Cited By ~ 1

Author(s):

Rosario Gajardo-Gómez ◽

Cristian A. Santibañez ◽

Valeria C. Labra ◽

Gonzalo I. Gómez ◽

Eliseo A. Eugenin ◽

...

Keyword(s):

Connexin 43 ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Purinergic Signaling ◽

Atp Release ◽

P38 Map Kinase ◽

Motor Deficits ◽

Pannexin 1 ◽

Channel Opening ◽

Wide Range

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1β/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

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A2A Receptor-induced Overexpression of Pannexin-1 Hemichannels Indirectly Mediates Adenosine Fibrogenic Actions in Subcutaneous Human Fibroblasts by Favoring ATP Release

10.21203/rs.3.rs-787071/v1 ◽

2021 ◽

Author(s):

Carina Herman-de-Sousa ◽

Maria Adelina Costa ◽

Rafaela Pedro Silva ◽

Fátima Ferreirinha ◽

Severino Ribeiro ◽

...

Keyword(s):

Inflammatory Mediators ◽

Connexin 43 ◽

Myofascial Pain ◽

Subcutaneous Tissue ◽

Human Fibroblasts ◽

Atp Release ◽

Collagen Production ◽

Pannexin 1 ◽

A2a Receptors ◽

Inflammatory Conditions

Abstract Disorganization of the subcutaneous tissue due to inflammation and fibrosis is a common feature in patients with myofascial pain. Dermal accumulation of adenosine favours collagen production by human subcutaneous fibroblasts (HSCF) via A2A receptors (A2AR) activation. Adenosine mimics the fibrogenic effect of inflammatory mediators (e.g. histamine, bradykinin), which act by promoting ATP release from HSCF via pannexin-1 (Panx1) and/or connexin-43 (Cx43) hemichannels. However, this mechanism was never implicated in the A2AR-mediated actions. NECA and CGS21680C, two enzymatically-stable A2AR agonists, increased Panx-1, but reduced Cx43, immunoreactivity in cultured HSCF. This effect was accompanied by increases in ATP release and collagen production by HSCF. Involvement of A2AR was verified upon blockage of NECA and CGS21680 effects with the selective A2AR antagonist, SCH442416. Inhibition of Panx1 hemichannels with probenecid also decreased ATP release and collagen production by HSCF under similar conditions. Superfluous ATP release by HSCF exposed to A2AR agonists overexpressing Panx1 hemichannels contributes to keep high [Ca2+]i levels in the presence of inflammatory mediators, like histamine. Adenosine A2AR-induced Panx1 overexpression was shown here for the first time; this feature indirectly implicates ATP release in the fibrogenic vicious cycle putatively operated by the nucleoside in subcutaneous tissue fibrosis and myofascial inflammatory conditions.

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Structure versus function: Are new conformations of pannexin 1 yet to be resolved?

The Journal of General Physiology ◽

10.1085/jgp.202012754 ◽

2021 ◽

Vol 153 (5) ◽

Author(s):

Carsten Mim ◽

Guy Perkins ◽

Gerhard Dahl

Keyword(s):

Structural Information ◽

Purinergic Signaling ◽

Atp Release ◽

Strong Support ◽

Decisive Role ◽

Chloride Ions ◽

Pannexin 1 ◽

Selective Membrane ◽

Selective Channel ◽

Structure Versus

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.

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Pannexin-1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

10.1101/380469 ◽

2018 ◽

Author(s):

Marco Tozzi ◽

Jacob B. Hansen ◽

Ivana Novak

Keyword(s):

Adipose Tissue ◽

Purinergic Receptors ◽

Purinergic Signaling ◽

Cell Metabolism ◽

Atp Release ◽

Extracellular Atp ◽

Adrenergic Stimulation ◽

Pannexin 1 ◽

White Adipocytes ◽

Obesity And Diabetes

One-sentence summaryInsulin inhibits ATP release in adipocytesAbstractExtracellular ATP signaling is involved in many physiological and pathophysiological processes, and purinergic receptors are targets for drug therapy in several diseases, including obesity and diabetes. Adipose tissue has crucial functions in lipid and glucose metabolism and adipocytes express purinergic receptors. However, the sources of extracellular ATP in adipose tissue are not yet characterized.Here, we show that upon adrenergic stimulation white adipocytes release ATP through the pannexin-1 pore that is regulated by a cAMP-PKA dependent pathway. The ATP release correlates with increased cell metabolism, and extracellular ATP induces Ca2+ signaling and lipolysis in adipocytes and promotes macrophages migration. Most importantly, ATP release is markedly inhibited by insulin, and thereby auto/paracrine purinergic signaling in adipose tissue would be attenuated. Furthermore, we define the signaling pathway for insulin regulated ATP release.Our findings reveal the insulin-pannexin-1-purinergic signaling cross-talk in adipose tissue and we propose that deregulation of this signaling may underlie adipose tissue inflammation and type-2 diabetes.

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