Kenji F. Shoji

Modulation of Brain Hemichannels and Gap Junction Channels by Pro-Inflammatory Agents and Their Possible Role in Neurodegeneration

In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process.

Amyloid β-Induced Death in Neurons Involves Glial and Neuronal Hemichannels

The mechanisms involved in Alzheimers disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-β peptide (Aβ) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aβ. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aβ25–35 increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aβ. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aβ25–35-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aβ25–35-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aβ leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aβ.

Cell membrane permeabilization via connexin hemichannels in living and dying cells

Vertebrate cells that express connexins likely express connexin hemichannels (Cx HCs) at their surface. In diverse cell types, surface Cx HCs can open to serve as a diffusional exchange pathway for ions and small molecules across the cell membrane. Most cells, if not all, also express pannexins that form hemichannels and increase the cell membrane permeability but are not addressed in this review. To date, most characterizations of Cx HCs have utilized cultured cells under resting conditions have and revealed low open probability and unitary conductance close to double that of the corresponding gap junction channels. In addition, the cell membrane permeability through Cx HCs can be markedly affected within seconds to minutes by various changes in the intra and/or extracellular microenvironment (i.e., pH, pCa, redox state, transmembrane voltage and intracellular regulatory proteins) that affect levels, open probability and/or (single channel) permeability of Cx HC. Net increase or decrease in membrane permeability could result from the simultaneous interaction of different mechanisms that affect hemichannels. The permeability of Cx HCs is controlled by complex signaling cascades showing connexin, cell and cell stage dependency. Changes in membrane permeability via hemichannels can have positive consequences in some cells (mainly in healthy cells), whereas in others (mainly in cells affected by acquired and/or genetic diseases) hemichannel activation can be detrimental.

Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential

Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca2+ and enhanced by low Ca2+, positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La3+, by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeant-reducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.

Glucose increases intracellular free Ca2+ in tanycytes via ATP released through connexin 43 hemichannels

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal‐glial cells, termed tanycytes. The latter express glucose‐sensing proteins, including glucose transporter 2, glucokinase, and ATP‐sensitive K+ (KATP) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose‐induced rise of intracellular free Ca2+ concentration ([Ca2+]i) in cultured β‐tanycytes was examined. Fura‐2AM time‐lapse fluorescence images revealed that glucose increases the intracellular Ca2+ signal in a concentration‐dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a KATP channel‐dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y1 receptors followed by inositol trisphosphate receptor activation and Ca2+ release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca2+]i in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.

Connexin Hemichannel Composition Determines the FGF-1–induced Membrane Permeability and Free [Ca2+]i Responses

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.

Possible involvement of different connexin43 domains in plasma membrane permeabilization induced by ischemia-reperfusion

In vitro and in vivo studies support the involvement of connexin 43-based cell-cell channels and hemichannels in cell death propagation induced by ischemia-reperfusion. In this context, open connexin hemichannels in the plasma membrane have been proposed to act as accelerators of cell death. Progress on the mechanisms underlying the cell permeabilization induced by ischemia-reperfusion reveals the involvement of several factors leading to an augmented open probability and increased number of hemichannels on the cell surface. While open probability can be increased by a reduction in extracellular concentration of divalent cations and changes in covalent modifications of connexin 43 (oxidation and phosphorylation), increase in number of hemichannels requires an elevation of the intracellular free Ca2+ concentration. Reversal of connexin 43 redox changes and membrane permeabilization can be induced by intracellular, but not extracellular, reducing agents, suggesting a cytoplasmic localization of the redox sensor(s). In agreement, hemichannels formed by connexin 45, which lacks cytoplasmic cysteines, or by connexin 43 with its C-terminal domain truncated to remove its cysteines are insensitive to reducing agents. Although further studies are required for a precise localization of the redox sensor of connexin 43 hemichannels, modulation of the redox potential is proposed as a target for the design of pharmacological tools to reduce cell death induced by ischemia-reperfusion in connexin 43-expressing cells.

Gap Junctions at the Dendritic Cell-T Cell Interface Are Key Elements for Antigen-Dependent T Cell Activation

The acquired immune response begins with Ag presentation by dendritic cells (DCs) to naive T cells in a heterocellular cell-cell contact-dependent process. Although both DCs and T cells are known to express connexin43, a gap junction protein subunit, the role of connexin43 on the initiation of T cell responses remains to be elucidated. In the present work, we report the formation of gap junctions between DCs and T cells and their role on T cell activation during Ag presentation by DCs. In cocultures of DCs and T cells, Lucifer yellow microinjected into DCs is transferred to adjacent transgenic CD4+ T cells, only if the specific antigenic peptide was present at least during the first 24 h of cocultures. This dye transfer was sensitive to gap junction blockers, such as oleamide, and small peptides containing the extracellular loop sequences of conexin. Furthermore, in this system, gap junction blockers drastically reduced T cell activation as reflected by lower proliferation, CD69 expression, and IL-2 secretion. This lower T cell activation produced by gap junction blockers was not due to a lower expression of CD80, CD86, CD40, and MHC-II on DCs. Furthermore, gap junction blocker did not affect polyclonal activation of T cell induced with anti-CD3 plus anti-CD28 Abs in the absence of DCs. These results strongly suggest that functional gap junctions assemble at the interface between DCs and T cells during Ag presentation and that they play an essential role in T cell activation.

Regulation of Hemichannels and Gap Junction Channels by Cytokines in Antigen-Presenting Cells

Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.

ATP Is Required and Advances Cytokine-Induced Gap Junction Formation in Microglia In Vitro

Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-α/IFN-γ-induced dye coupling, probably through the induction of IL-1β release. Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.