Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology, and roles

1For a long time, it has been taken as dogma that connexin (Cx) proteins can only function as gap junction channels. Indeed, before the aggregation of Cxs at the junctional plaque and subsequent formation of gap junctions, hexameric rings of Cxs, termed connexons, were initially assumed to remain closed. An obvious reason for this occlusion was that, as gap junction channels are “poorly” selective for ions and permeable to low molecular weight molecules (<1 to 1.2 KDa), if once at the membrane connexons could open, the cell would lose its integrity or at least would have to spend substantial energy to maintain this energetically unfavorable condition. Such statements began to be challenged when evidence for “functional hemichannels,” a term proposed to substitute for plasma membrane connexons, were reported in the early 1990s. In these pioneering studies, Cx hemichannels were opened either by large depolarization in Xenopus oocytes or by lowering the extracellular calcium ion (Ca2+) concentration in horizontal cells. Later on, the occurrence of functional hemichannels (i.e., hemichannels that can be turned into the open state) composed of Cx43 was demonstrated in primary cultures of astrocytes in the absence of external Ca2+. This observation was followed by the demonstration that metabolic inhibition performed in the presence of normal external Ca2+ concentrations (1–2 mM) induced cell permeabilization, due to Cx43 hemichannel opening, before loss of membrane integrity. More recently, hemichannel opening in astrocytes was triggered either by treatment with pro-inflammatory cytokines or selective lipopolysaccharide (LPS) stimulation of microglia co-cultured with astrocytes, again in the presence of external Ca2+. The opening of Cx43 hemichannels in astrocytes was also demonstrated to occur in experiments designed to decipher the mechanism of intercellular Ca2+ wave propagation in astrocytes to which both gap junction channels and hemichannels contribute. In this case, the release of ATP through Cx43 hemichannels turned out to play a key role in Ca2+ wave propagation mediated by an extracellular paracrine signaling component. All these initial experiments on Cx hemichannels utilized astrocytes as a cell model giving to these cells new roles in neuroglial interaction.

2At the turn of the century another membrane channel family, the pannexins (Panxs), was identified and demonstrated to be orthologs of innexins, the gap junction proteins expressed in invertebrates. So far, there are no reports of gap junctional communication supported by native Panxs, although over expression of Panx1 enhances gap junctional coupling in glioma cells. They form membrane channels contributing to membrane permeabilization, similar to Cx hemichannels, and they are expressed in glial cells. The aim of the present review is to summarize what is actually known about Cx hemichannels and Panx channels in glia (mainly astrocytes and microglia) and to discuss their contribution to neuroglial interactions taken in the normal and pathological context. An overview of methods and approaches to investigate Cx hemichannels and Panx channels has recently been published; we refer the reader to this paper for a more detailed discussion of methodological aspects.

3Gap junctions provide a unique direct conduit between cells, influencing crucial cellular processes through activities including electrical conductance and metabolic cooperation, mediated by the passage of ions, amino acids, glucose, glutathione, ATP, and small signaling molecules (reviewed in ), as well as microRNAs. When one considers Cx expression in the brain, it is evident that they are most abundant in glia, namely the astrocytes, oligodendrocytes, and microglia. With the genomic characterization of Cx and Panx gene families, it has been determined that there are at least 10 Cxs and 2 Panxs expressed in the brain (see below). There is also a temporal (i.e., developmental) and spatial (i.e., cell type and brain region) pattern regarding this expression and localization. Furthermore, these channel proteins can have different roles in different cell types, either as the canonical gap junction intercellular channel or as a hemichannel (; Figure 1). Cx hemichannels and Panx1 channels mediate the release of ATP that activates P2 receptors, promoting rises in intracellular Ca2+ concentrations; gap junction channels mediate the intercellular transfer of second messenger propagated calcium signal. Under pathological conditions, Panx1 channels have been proposed to mediate activation of the inflammasome both in neurons and astrocytes, whereas Cx43 hemichannels are involved in acceleration of astroglia and neuronal cell death triggered by hypoxia-reoxygenation in high glucose and β-amyloid peptide, respectively. A further level of complexity can be envisioned in the context of Cxs, where the heteromeric assembly of different Cx proteins into a single channel can result in distinct differences in biophysical properties. This feature is also true for Panxs since Panx1 can form both homomeric and heteromeric channels with Panx2. Thus for specific cellular functions, the complement of Cxs and Panxs expressed is important in the physiological or pathological context being considered. While other “non-channel” functions are emerging for Cxs and Panxs, this review will be limited to channel-related functions and properties.

4While this review will focus on gap junction channels and hemichannels in brain glial cells, it is important to note that gap junction proteins have also been identified in brain neurons. These include Cx26, Cx30.2, Cx36, Cx45, and Cx57. Neurons also express Panx1 in vivo, and recently it has been reported that neural progenitors in the adult rodent hippocampus express Panx2. It is thus possible, and has been reported in some situations, that neurons form gap junctions with astrocytes since some of the same Cxs are expressed in both cell types. Astrocytes express multiple Cxs, including Cx43, Cx26, Cx30, Cx40, Cx45, and Cx46, as well as Panx1 and Panx2. Non-treated microglia express Cx43, Cx36, and Cx32, as well as Panx1. Finally, oligodendrocytes express Cx32, Cx47, and Cx29 in vivo. The complexity of Cxs and Panxs functioning as gap junction channels/hemichannels and channels, respectively, in these various cell types can be viewed as providing a background level of activity to support and modulate the complex neural activity mediated via gliotransmission.

5The expression profiles noted have been associated with mature neurons and glial cell types. However, these various cell types arise developmentally from neuronal and glial precursors. Several studies have demonstrated changes in expression of Cxs and Panxs during brain development (reviewed in; ). Given that several reports have described disturbed neural development when expression of some Cxs is modified through the use of gene knockdown or knockout strategies, gap junction channels and hemichannels are likely to play significant roles in processes including cell proliferation, migration, and differentiation.

6Initial studies on Cx functions in neural cells in culture and in brain slices focused on their expected role in forming gap junction channels, confirming their function via electrophysiological analysis or methods using low molecular weight dye tracers, which can pass from cell to cell through these intercellular channels. Therefore, many of the phenotypic changes observed when enhancing or decreasing Cx expression were attributed to changes in gap junctional communication. With the recognition that Cxs can also form hemichannels, the consequences of Cx expression began to be considered in a new way, and appropriate methods developed to assess their presence and activity. Cx hemichannels and Panx channels are more typical of most membrane channels, like ion channels, in their direct communication between intracellular and extracellular space. However, they are unique in their pore size and conductance properties.

7The formation of gap junction channels and hemichannels by Cxs can help explain some contradictory effects of altering Cx expression. For example, enhanced Cx43 expression in brain glial cells, in culture and in vivo, has been shown to be both neuroprotective and neurodestructive in ischemic and excitotoxic situations (reviewed in; ). Recent studies have indicated that gap junction channels formed by Cx43 can be neuroprotective, while Cx43 hemichannels may be more detrimental to neuronal survival. The following sections of this review provide further insight into the distinct properties of hemichannels and how these impact the progression of neurological disorders.

8Today, most Cxs expressed in rodent glial cells, including Cx26, Cx30, Cx32, Cx43, and Cx45, have been shown to form functional hemichannels in endogenous and/or exogenous expression systems. Nevertheless, it remains unknown if this is also the case for Cx29 and Cx47 found in oligodendrocytes. In exogenous expression systems, Cx30, Cx32, Cx43, and Cx45 hemichannels show a very low open probability at resting membrane potential and normal concentration of the extracellular divalent cations, Ca2+ and Mg2+. For Cx26 hemichannels, these properties are species-specific; human and sheep but not rat Cx26 forms hemichannels that at resting membrane potential and normal concentration of extracellular divalent cations show opening activity but the rat protein is characterized by a single evolutionary amino acid change (D159N) that limits this feature.

9Opening of Cx hemichannels studied so far can be promoted by positive transmembrane voltages and reduced concentrations of extracellular divalent cations. These conditions have been most frequently used to gain knowledge on biophysical features of hemichannels, including permeability properties (see below), identification of voltage sensitivity, unitary conductance, kinetic properties, and polarity of closure. However, other gating mechanisms can also promote opening of hemichannels, as described below (see Regulation). In general, hemichannels show a time- and voltage-dependent macroscopic current, activating with depolarization and inactivating with hyperpolarization. However, this feature is not equal in hemichannels formed by different Cxs. For example, the voltage dependency is strong for mouse Cx30 hemichannels but detectable only at very high positive potentials for rat Cx43 hemichannels. For Cx32 hemichannels, the conductance is determined by charges dispersed over the pore pathway. Hemichannels formed by human Cx26 or rat Cx43 show bipolar voltage behavior; however, this appears to be less well established for Cx43, for which unitary current events were found to monotonically rise in the voltage range of +30 to +90 mV. Hemichannels present two distinct voltage-gating mechanisms, which are called loop- (slow) and V(j)- (fast) gating, respectively. For Cx32 hemichannels the loop-gate is mediated by a conformational change that reduces the pore diameter in a region located close to the first transmembrane domain and first extracellular loop. It was recently also proposed that the intracellular pore entrance narrows from 15 to 10 Å with loop-gated but not with V(j)-gated channel closure. Moreover, it was shown that the extracellular entrance does not undergo evident large conformational changes with either voltage-gating mechanisms. The unitary conductance is characteristic for hemichannels with specific Cx composition; the fully open state for Cx26 hemichannels is characterized by ~320 pS unitary conductance, for Cx32 hemichannels this is ~90 pS, for Cx43 hemichannels ~220 pS and for Cx45 hemichannels ~57 pS. They also present conductance substates as a result of an inactivation mechanism that hinders complete activation of hemichannels under prolonged depolarization. The most general features mentioned above have been useful to demonstrate and understand the involvement of hemichannels in different physiologic functions and also to identify hemichannels with specific Cx composition in glial cells. However, much more remains to be investigated since Cxs are post-translationally modified and thus, their biophysical features might be affected according to the metabolic state of the cell.

10The biophysical properties of Panx channels are much less documented than those of Cx hemichannels, possibly as a result of their more recent discovery. Panx1 channels have been so far demonstrated to be present in microglia, astrocytes, and oligodendrocytes, whereas Panx2 is expressed in post-ischemic astrocytes. The open state of Panx1 channels is promoted by positive membrane voltages and elevation of intracellular free Ca2+ concentration. Voltage clamp experiments have indicated widely divergent unitary conductances for Panx1 channels, varying from 68 to 550 pS. This controversy in the single channel conductance of Panx1 channels has not been resolved yet and might be explained in part by differences in recording conditions or solutions, cell type, phenotypic differences, and post-translational modifications. It could be partially explained by simultaneous opening of channels and thus, high conductance values might represent multiples of a single channel with a lower conductance value. An issue to keep in mind is that biophysical properties of exogenously expressed Panx channels might be different from those expressed simultaneously with all membrane and intracellular proteins (e.g., P2X receptors) that might interact with them in endogenous expression systems.

11In addition to selective ion channels or transporters, other channels can alter the permeability of the cell membrane under physiological and pathological conditions. The origin of these permeability changes could be Cx- and Panx-based channels located at the cell surface. However, other membrane channels might also be involved in drastic changes in membrane permeability [e.g., P2X7 receptors, transient receptor potential (TRP) channels and calcium homeostasis modulator 1 (CALHM1) ion channel] and their possible involvement should be proved or disproved when testing the permeability of Cx hemichannels or Panx channels in a particular cell type.

12Net changes in membrane permeability due to hemichannels have been most frequently studied through the use of inhibitors or molecular biology approaches that induce or abolish the expression of Cxs or Panxs. With these experimental approaches it has been proposed that hemichannels (Panx1 and/or Cx26, Cx32, and Cx43) allow the release of precursor or signaling molecules [e.g., including NAD+, ATP, adenosine, inositol trisphosphate (IP3), and glutamate;;;;;;;;; ], uptake of second messengers [e.g., cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), nitric oxide (NO), and Ca2+;;,;;;; ) and uptake of various metabolites (e.g., glucose, glutathione, and ascorbate;;;;; see Table 1 for Cx43 hemichannels and Panx1 channels). In addition, it has been demonstrated that Cx hemichannels are permeable to synthetic molecules [e.g., Lucifer yellow, ethidium, calcein, propidium, 5(6)-carboxyfluorescein, and 2-(4-amidinophenyl)-1H-indole-6-carboxamide (DAPI); for review, see ]. The fact that Cx26 and Cx32 hemichannels reconstituted in lipid membranes are permeable to Ca2+ and cyclic nucleotides, respectively, indicates that no additional binding partners or associated molecules are required for hemichannels to function as conduits for small molecules or ions across the cell membrane. Since the membrane permeability change due to transient hemichannel opening may allow release of enough molecules to reach effective concentrations in a confined extracellular space, astroglial hemichannels have been proposed as membrane pathways for paracrine/autocrine cell signaling with physiological and pathological implications for glial and neuronal cells.

13Membrane permeability changes via Cx hemichannels are in part explained by variations in the number of hemichannels available at the cell surface. With regard to this, an increase in the number of surface Cx43 or Cx45 hemichannels explains the increase in membrane permeability induced by fibroblast growth factor-1 (FGF-1) in cells that express these proteins. However, in other conditions such as exposure to extracellular solution without divalent cations or tanycytes treated with extracellular glucose, the increase in membrane permeability is explained by opening of hemichannels via a gating mechanism. In both cases there is a net increase in membrane permeability mediated by hemichannels, possibly without changes in intrinsic permeability properties of hemichannels themselves. Nonetheless, changes in membrane permeability could also result from alterations in permeability features of hemichannels. For instance, protein kinase C (PKC)-mediated phosphorylation of the six subunits of Cx43 hemichannels reconstituted in lipid membranes reduces the pore permeability allowing permeation of the small hydrophilic solute ethylene glycol (Mr 62) but not sucrose (Mr 342; ). Changes in other hemichannel permeability properties, such as affinity or charge selectivity induced by covalent modification including phosphorylation by other protein kinases or nitrosylation, cannot be ruled out yet.

14The permeability properties depend on the size, shape, and net charge of each permeant ion or molecule while the concentration gradient across the cell membrane acts as the driving force. Since the study of permeability characteristics of a membrane transporter should be done with a rapid time scale (within a few seconds to a minute) it is important to have a library of permeant molecules that allow real time determination of their disappearance or appearance. Transport via Cx43 hemichannels has been shown to be cooperative and saturable with parameters (e.g., Vmax, Km, and Hill coefficient) depending on the permeant cationic species. These findings imply that opening of hemichannels does not necessarily lead to loss of important intracellular molecules because of competition effects with other, less important molecules or ions and this interpretation might be valid to transport in both directions across the cell membrane. Of note, the permeability properties of hemichannels to anionic molecules have not been reported yet. To the best of our knowledge, the permeability properties of Panx1 channels remain equally unknown. It is also not known whether channels formed by different Cxs or Panxs show different permeability properties such as size exclusion and charge selectivity. In general, quantitative kinetic properties of permeation of Cx hemichannels and Panx channels remain largely unexplored. Table 1 summarizes current evidence on the permeability of Cx43, Cx32 hemichannels, and Panx1 channels in brain glial cells.

15Hemichannels are regulated by diverse conditions of the extracellular and intracellular microenvironments. They include Ca2+ concentration outside and inside the cell, monovalent cation concentration, pH, mechanical stress, extracellular ligands, protein kinases, protein phosphatases, as well as oxidant and reducing agents. Here, the most recent studies not included in previous reviews, will be presented.

16Unapposed Cx hemichannels of cells in culture are preferentially closed but can be opened by several trigger conditions (reviewed in, ). These include, transmembrane voltage with activation threshold of -30 mV for Cx30 and +50 mV for Cx43 in the presence of normal extracellular Ca2+, mechanical forces/strain, a decrease of extracellular Ca2+, an increase of intracellular free Ca2+, alterations in phosphorylation status and redox status, and ischemia-mimicking conditions.

17Role of extracellular Ca2+. A decrease of extracellular divalent cation concentration was one of the first conditions reported to stimulate Cx43 hemichannel opening, as introduced above. Work in a hepatoma cell line expressing Cx43 demonstrated that a decrease of the extracellular Ca2+ concentration triggered the uptake of Lucifer yellow, a hemichannel-permeable fluorescent dye. Similar observations were obtained in astrocytes, where hemichannel opening was demonstrated to trigger the release of glutamate and aspartate. Lowering extracellular Mg2+ appeared to promote low Ca2+-triggered hemichannel opening but Ca2+ appeared to be the dominant factor. It is thought that normal physiological extracellular Ca2+ concentrations (~1.8 mM) keep non-junctional Cx hemichannels closed in the plasma membrane, until they become incorporated into gap junction channels where they open by a process called “loop gating” as a consequence of extracellular loop interactions. In general, the half-maximal effective concentration for Ca2+-related opening of Cx43 hemichannels is in the order of 100 μM extracellular Ca2+. The proposed mechanism of millimolar Ca2+ closure of Cx hemichannels involves an interaction of extracellular Ca2+ with a Ca2+ binding site that consists of a ring of aspartate residues at the extracellular vestibule of the hemichannel pore thereby closing the channel. Such a mechanism has been best documented for Cx32 but appears to be also plausible for the astroglial Cxs, Cx30 and Cx43. Recently, have elegantly exploited the extracellular Ca2+-sensitivity of astroglial hemichannels to trigger their opening by photo-activating a Ca2+ buffer (thereby increasing its Ca2+ affinity) added to the interstitial fluid of a hippocampal acute slice preparation (discussed further below under Section “Impact of Hemichannel-Mediated Gliotransmission on Synaptic Activity and Behavior”).

18Role of intracellular Ca2+. Besides extracellular Ca2+, intracellular cytoplasmic Ca2+ also influences hemichannel function. In fact, it was found that Cx hemichannel opening triggered by a decrease of extracellular Ca2+ could be inhibited by ester-loading the cells with the Ca2+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), added to dampen intracellular Ca2+ changes. Further investigations demonstrated that Cx32 hemichannels are activated by moderate (below 500 nM) changes in intracellular Ca2+ and inhibited by Ca2+ changes above 500 nM. Subsequent work showed that Cx43 hemichannels are also characterized by such a biphasic, bell-shaped, response to changes in intracellular Ca2+ concentration, in ATP release hemichannel studies as well as in dye uptake studies in glioma cells and other cell types. Recently, single channel studies of Cx43 hemichannels robustly confirmed these observations. Cx hemichannels are Ca2+ permeable and the biphasic dependency on intracellular Ca2+ thus confers a positive feedback below 500 nM intracellular Ca2+ concentration (Ca2+-induced Ca2+ entry via Cx43 hemichannels) and negative feedback at higher concentrations. Interestingly, IP3 receptor channels present in the endoplasmic reticulum (ER) are Ca2+ release channels that have a similar bell-shaped Ca2+ dependency. Positive and negative Ca2+ feedback at IP3 receptor channels forms the basis of Ca2+ oscillations, thus, it was hypothesized that Ca2+ feedback at Cx hemichannels could also play a role in Ca2+ oscillations. Experimental work with various peptides targeting Cx hemichannels indeed demonstrated that inhibiting hemichannel opening, or preventing their closure in response to high intracellular Ca2+, blocked Ca2+ oscillations triggered by bradykinin. Thus, Cx hemichannels constitute a putative gliotransmitter release pathway. Also, they may contribute to Ca2+ oscillations in astrocytes and thereby potentially lead to gliotransmitter release via other Ca2+-dependent pathways. In brain endothelial cells, it was found that Cx hemichannel blocking peptides prevented the opening of the blood–brain barrier, triggered by bradykinin, by inhibiting Ca2+ oscillations in the endothelial cells. Below, we discuss in more detail some peptide-based hemichannel inhibitors.

19Elevations in extracellular K+ shift the activation potential of Panx1 hemichannels to a more physiological range. Panx1 hemichannels expressed in astrocytes might serve as K+ sensors for changes in the extracellular milieu such as those occurring under pathological conditions. This type of regulation might also occur under physiological conditions with high neuronal activity. Thus, elevations in extracellular K+ of the magnitude occurring during periods of high neuronal activity have been proposed to affect the intercellular signaling among astrocytes. Accordingly, the gliotransmitter release in the hippocampus in response to high neuronal activity is sensitive to P2 receptor and Panx1 channel blockers. Cx30 gap junction channels have recently been demonstrated to be regulated by the concentration of external K+; whether astroglial Cx hemichannels are influenced by the extracellular K+ concentration is currently not known.

20Under pathological conditions, the intracellular and extracellular pH may display abrupt changes. Related to this issue and in the presence of extracellular divalent cations, Cx43 hemichannels are opened by alkaline pH applied at the extracellular side and preferentially closed at physiologic pH. The activity of Cx45 hemichannels recorded at positive membrane potentials and in the absence of extracellular Ca2+ is drastically reduced by acidification. In addition, the pH sensitivity might be potentiated by protonated aminosulfonates, such as taurine that could be in millimolar concentration in the cytoplasm of several cell types including astrocytes. This effect is Cx specific; Cx26 but not Cx32 hemichannels are closed by aminosulfonates at constant pH. At the ultrastructural level (high-resolution atomic force microscopy), Cx26 hemichannels are closed at pH < 6.5 [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer] and opened reversibly by increasing the pH to 7.6. Moreover, molecular studies have revealed that Cx26 hemichannel closure induced by taurine requires the interaction between the cytoplasmic loop and the C-terminal (CT; ).

21Mechanical stress is another condition that might be relevant under pathological situations. For example, after brain trauma the extracellular medium becomes hypertonic due to the release of intracellular solutes to the extracellular microenvironment. In astrocytes, hypertonicity induces glutamate release through Cx43 hemichannels. This response could be mediated by integrin α5β1, a RhoA GTPase and the contractile system. This sensitivity is likely to result from the interaction between negatively charged amino acid residues of the CT end (Asp278 and Asp279) and a domain of the intracellular loop. Activation of Panx1 channels by mechanical stress is not present in all cell types, suggesting that it should be tested in glial cells in order to validate its possible relevance in these cells.

22The functional state of glial Cx hemichannels and Panx1 channels is actively regulated by extracellular signals. In primary cultures of mouse astrocytes, stomatin inhibits Panx1-mediated whole cell currents by interacting with its CT. In contrast, pro-inflammatory agents such as the β-amyloid peptide increase the surface expression and activity of Cx43 hemichannels and Panx1 channels in microglia and Cx43 hemichannels in astrocytes. Moreover, in brain astrocytes, epidermal growth factor (EGF) and FGF-2 inhibit Cx hemichannel activity via the mitogen-activated protein kinase cascade, and the effect of the growth factors is reversed by interleukin-1β (IL-1β; ). In contrast, pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and IL-1β] released by LPS-activated microglia increase the activity of astroglial Cx43 hemichannels via p38 kinase and this response is inhibited by activation of CB1 receptors by synthetic cannabinoid agonists. The orchestrated involvement of Cx hemichannels and Panx1 channels has also been found in other experimental preparations. In spinal cord astrocytes, FGF-1 leads to vesicular ATP release followed by sequential activation of Panx1 and Cx43 hemichannels. Moreover, up-regulation of astroglial Panx1 channels and Cx43 hemichannels has been found in brain abscess.

23Cx43 hemichannels expressed by tanycytes, glial cells present in the hypothalamus, open in seconds after exposure to physiological concentrations of extracellular glucose. This response is mediated by the sequential activation of glucosensing proteins, a response that is absent in cortical astrocytes. However, high glucose levels enhance the increase in astroglial Cx43 hemichannel activity induced by hypoxia-reoxygenation.

24As a result of cell interaction with extracellular signals or conditions that affect the energy supply, intracellular metabolic pathways involving levels of free Ca2+ concentration, activity of protein kinases and phosphatases, as well as levels or activity of intracellular redox molecules, are affected. Therefore, drastic reduction in ATP levels are expected to favor the dephosphorylated state of Cx43 hemichannels and thus, they could present an increased activity. Moreover, the lack of energy leads to a rise in intracellular free Ca2+ that favors the generation of reactive oxygen and nitrogen species including NO. The latter induce rapid nitrosylation of Cx43 and opening of hemichannels. This mechanism might partially explain the increase in Cx43 hemichannel activity observed in astrocytes under ischemia-like conditions since free radical scavengers, dithiothreitol, a sulfhydryl (SH) reducing reagent, and high intracellular levels of reduced glutathione revert the hemichannel activity to levels found in normal cells. Finally, extracellular ATP mediates the ischemic damage to oligodendrocytes and is partially explained by the activation of Panx1 channels. In contrast to Cx43 hemichannels, S-nitrosylation of Panx1 channels reduces their activity, suggesting that their opening might be relevant only under conditions where NO is overcome by -SH reducing agents such as reduced glutathione.

25Specifically targeting unapposed hemichannels is difficult because they are composed of the same Cx building blocks as gap junction channels. As a consequence, most gap junction blockers also inhibit hemichannels (see ). Moreover, many Cx hemichannel/gap junction channel blockers also block Panx channels. Gap junction blockers include glycyrrhetinic acid and its derivative carbenoxolone, long-chain alcohols like heptanol or octanol, halogenated general volatile anesthetics like halothane, fatty acids like arachidonic acid and oleic acid, fatty acid amides like oleamide and anandamide, and fenamates like flufenamic acid, niflumic acid, or meclofenamic acid (reviewed in ). Most of these substances have been used in the past to block hemichannels but the results obtained can only be unequivocally interpreted in terms of hemichannels when the contribution of gap junction channels is negligible or absent. Substances like the trivalent ions gadolinium (Gd3+) and lanthanum (La3+) block hemichannels without inhibiting gap junction channels or Panx1 channels, but these ions also inhibit other channels including maxi-anion channels and Ca2+ channels. A possible way to differentiate between Cx hemichannels and Panx channels is the use of long-chain alcohols that block Cx hemichannels but have only a very small effect of Panx channels. In contrast, low concentration (5–10 μM) of carbenoxolone blocks Panx1 channels and only has a minor inhibitory effect on Cx hemichannels. However, all these compounds are not specific and in addition to their inhibitory effects on Cx and Panx channels (including gap junctions channels and hemichannels) they also affect other neuronal and glial membrane properties which limits their use (see ).

26A more specific targeting of Cx channels is to be expected, at least in principle, from mimetic peptides of Cx proteins. Peptides identical to certain sequences on the Cx protein have been extensively used to interfere with the Cx channel function (Figure 2). The first Cx mimetic peptides that were introduced in the 1990s were identical to specific domains on the extracellular loops (first or second extracellular loop) of the Cx protein. The sequences mimicked were located in domains thought to be involved in the docking of two hemichannels during formation of a full gap junction channel. Exogenous addition of peptides mimicking parts of these domains were hypothesized to interact with yet unknown extracellular loop domains, thereby preventing extracellular loop interactions of apposed hemichannels during docking and thus hindering gap junction channel formation. These domains, known as Gap26 and Gap27, are present on the first and second extracellular loops, respectively. Accordingly, Gap26 and Gap27 peptides were indeed found to inhibit gap junctional coupling. Interestingly, subsequent work demonstrated that Gap26 and Gap27 peptides also inhibited unapposed hemichannels with which these peptides are supposed to interact. Currently, clear evidence that these peptides indeed interact with the extracellular loops is only available for Gap26. Nevertheless, both Gap26 and Gap27 rapidly inhibit hemichannels within minutes, followed by a somewhat delayed inhibition of gap junction channels, often in the range of hours, with some exceptions. The exact mechanism of hemichannel block is currently not known. It is known that Gap26/27 influence voltage-dependent gating but this does not explain their inhibitory action on hemichannels opened by triggers other than voltage. It has been suggested that these peptides inhibit hemichannels by just blocking the pore because of steric hindrance effects. However, this has been carefully checked and it was found that this only occurs at very high (1 mM and above) concentrations. Although Gap26/27 peptides proved to be interesting tools to inhibit Cx hemichannels, their delayed inhibition of gap junction channels remains a serious drawback. Moreover, Gap26/27 peptides also show rather limited specificity toward different Cx types. Indeed, the extracellular loop domains mimic sequences that show pronounced homology between different Cx proteins. Thus, Gap27 directed against Cx43 in astrocytes is also known to inhibit channels or hemichannels composed of Cx37, a vascular Cx present in brain blood vessels.

27Besides peptides, antibodies have also been used to target the free extracellular loops of unapposed Cx hemichannels resulting in their inhibition. These tools offer additional advantage because they allow both functional blocking of the hemichannels and visualizing their distribution. However, currently single channel electrophysiological data showing hemichannel block by an antibody directed against an extracellular epitope is only demonstrated for Cx43 hemichannel activity induced in β-amyloid-treated astrocytes. Additionally, the specificity toward effects on other Cxs or Panxs still needs to be determined.

28To improve specificity toward different Cxs, peptides have been devised that should target sequences on the intracellular domains of the Cx protein. The intracellular sequences are poorly conserved and very different between different Cxs. The L2 peptide is identical to a sequence on the cytoplasmic loop of Cx43. This peptide is known to prevent the closure of gap junction channels by binding to the CT tail, thereby preventing interaction of the CT tail with the cytoplasmic loop of Cx43 where the L2 domain is located (; and references therein). Conceptually, interaction between the CT tail and the L2 domain is thought to block the gap junction channel pore upon exposure to acidification according to a particle-receptor model, and exogenous addition of L2 peptide prevents this interaction and its associated channel closure. Unexpectedly, it was found that the L2 peptide inhibited Cx43 hemichannels, based on ATP release studies, indicating that this peptide has distinct effects on unapposed hemichannels and those incorporated into gap junctions. Further work with Gap19, a mimetic nonapeptide located within the Cx43 L2 domain, demonstrated a similar action; it inhibited single channel hemichannel currents and ATP release via Cx43 hemichannels while it did not influence gap junctional coupling after 30 min and stimulated coupling after 24 h. Moreover, the effect appeared to be specific for Cx43 as Gap19 had no effect on Cx40 hemichannels or Panx1 channels. While the effects of L2 and Gap19 are understood at a molecular operational scale, the full details of why preventing interactions between the CT tail and the cytoplasmic loop have such different effects on hemichannels as compared to gap junction channels still needs further clarification. However, distinct effects on hemichannels and gap junction channels have been reported for other Cx channel influencing molecules or conditions; for example, basic FGF (bFGF) and cytokines like TNF-α or IL-1β have all been demonstrated to promote Cx43 hemichannel function while inhibiting gap junctions. Given the specific actions of L2 and Gap19 peptides at the level of both Cx types and channel types (hemichannels versus gap junction channels), these peptides offer promising possibilities to explore the role of astroglial Cx43 hemichannels in the complex environment of intact neural tissues, ex vivo or in vivo. Recent work has, for example, demonstrated that injection of L2 peptide in the rat amygdala strongly suppresses fear memory (; discussed further below under Section “Impact of Hemichannel-Mediated Gliotransmission on Synaptic Activity and Behavior”). Equally interesting with respect to identifying the role and functions of Cx43 hemichannels is the G138R mutation of Cx43 in which a Gly at position 138 is replaced by an Arg. This is one of the several mutants found in the inherited human disease oculodentodigital dysplasia (ODDD). Importantly, G138R mutant Cx43 is characterized by a loss-of-function of gap junctional coupling and a gain-of-function of hemichannels. Thus, this particular mutant Cx43 can be used to stimulate Cx43 hemichannel function as a complementary experimental approach to hemichannel inhibition. Recently, have exploited such an approach to investigate the role of hemichannels in glial–neuronal communication.

29Initially, gap junction channels were demonstrated to constitute the pathway allowing the propagation of intercellular Ca2+ signaling between astrocytes, termed as Ca2+ waves. Then, after the discovery of an external component that also contributes to this interglial signaling process, the involvement of Cx43 hemichannels and ATP release was identified. There is a consensus on the interpretation of these two sets of in vitro studies and it is admitted that both Cx channel functions are contributing. Indeed, they likely participate in various proportions to Ca2+ waves depending on the brain structure, the mode of stimulation to trigger the waves, the age and the in vitro conditions. In addition, several of these points could also account for the alternative proposal claiming that in astrocytes the external component is not supported by Cx43 hemichannels but by Panx1 channels. This point of controversy remains to be clarified in the future although it is likely that both might participate in various proportions under different conditions.

30More recently, several reports have confirmed that Ca2+ waves in astrocytes are present in vivo in the normal brain as well as in pathological situations. As expected, the occurrence, amplitude and extent of in vivo Ca2+ waves are much more limited compared to the initial in vitro observations. However, as Ca2+ signaling is considered as the mode of cellular excitability in astrocytes, such waves certainly play a role in the spatial and temporal features of neuroglial interactions. As a consequence, this means that in many cases dynamic neuroglial interactions are not limited to the sole tri-partite synapse but also occur at a more integrated level involving larger cerebral areas. This situation is also likely true at the gliovascular interface where there is a high level of Cx expression between astroglial perivascular endfeet and where Ca2+ waves propagate from one initial endfoot to its neighbors. Such features might optimize the contribution of astrocytes to the control of the cerebral blood flow. However, while a role of Cx43 gap junction channels/hemichannels in pial arteriole dilation has been demonstrated in vivo for the glia limitans in response to sciatic stimulation, a similar mechanism remains to be investigated for perivascular astrocytes within the brain parenchyma.

31As indicated above, glial cells express several Cxs and out of these at least four of them (Cx26, Cx30, Cx32, and Cx43) have been demonstrated to form functional hemichannels, and at least Cx26, Cx32, and Cx43 serve as a membrane pathway for cell or liposome Ca2+ inflow. Of course, brief opening of Cx hemichannels could serve to transiently increase the intracellular Ca2+ concentration that could affect a broad variety of physiological cell functions. For example, Cx43 hemichannels contribute to maintain higher intracellular levels of free Ca2+ in cells treated with FGF-1 that proliferate and remain healthy. However, sustained hemichannel opening could promote deleterious results, including cell death, but the bell-shaped action of intracellular Ca2+ might prevent this ending process. Panx1 has also been detected in glial cells, as mentioned above. This glycoprotein has been shown to form channels in the ER permeable to Ca2+ but the permeability to Ca2+ of Panx1 channels present in the cell surface remains elusive. Moreover, direct demonstration of either Cx- or Panx-based channels as a Ca2+ membrane pathway in any glial cell type remains to be demonstrated.

32Through the so-called astrocyte–neuron lactate shuttle, astrocytes participate in an activity-dependent manner to the uptake of glucose from the blood supply and the delivery of lactate to neurons. In normal conditions, glucose and lactate transporters are the membrane elements that support this astroglial contribution to brain metabolism. While glucose is well known to be essential for correct brain function, its homeostasis is altered by inflammatory conditions. In vitro, an inflammatory situation for astrocytes can be mimicked either by co-culturing them with microglia selectively activated by the endotoxin LPS or by directly treating them with pro-inflammatory cytokines, such as TNF-α and IL-1β. Interestingly, in both cases these treatments induce an opposite regulation of Cx43 channels at least in cultured astrocytes: a decrease in gap junctional communication and an activation of hemichannels. In these conditions Cx43 hemichannels were shown to allow the influx of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a glucose fluorescent analog, demonstrating that reactive or inflamed astrocytes can take up more glucose via Cx43 hemichannels when their metabolic coupling through gap junction channels is reduced. Such opening of Cx43 hemichannels may represent an alternative metabolic pathway for glucose entry in astrocytes during pathological conditions associated with an inflammation status. Interestingly, this observation identifies a pathway that could contribute to the increase of glucose uptake found in several uncoupling situations; this was correlated with the up-regulation of GLUT-1 (glucose transporter 1) expression and to the induction of the expression of GLUT-3, an isoform that is not normally expressed in astrocytes, as well for type I and type II hexokinases, respectively. However, the respective contribution of hemichannels and glucose transporters in inflammatory conditions remains to be established. Because the permeability of the blood–brain barrier is increased during the inflammatory response, more glucose coming from the circulation could become available to the brain. Thus, in addition to transporters, the entry of glucose through Cx43 hemichannels could contribute to an increase of lactate formation by astrocytes that are adapted for anaerobic metabolism. Finally, the opposite regulation of Cx43 channels may lead to a failure in intercellular glucose trafficking that could be compensated for by the increase in glucose influx through Cx43 hemichannels in individual astrocytes. Moreover, although not demonstrated yet, Cx43 hemichannels could also support lactate efflux to feed neurons since Cx43 gap junction channels in astrocytes are permeable to lactate. As a whole, Cx43 hemichannel activation should certainly contribute to modify the metabolic status of reactive astrocytes, a situation that should be taken into account when considering the role of astroglia in brain inflammation.

33By analogy with the concept of a neurotransmitter, the term “gliotransmitter” has been proposed to name active molecules that are released by astrocytes and regulate neuronal activity as well as synaptic strength and plasticity. There are mainly three candidates that are proposed to play this role: glutamate, ATP, and D-serine. All of them can be released by a Ca2+-dependent vesicular release mechanism in astrocytes. However, at least glutamate and ATP have been demonstrated to permeate Cx43 hemichannels in astrocytes as well as Panx1 channels in microglia, thus Cx43 and Panx1 channels could also play a role in gliotransmission. Recently, the activation of Cx43 hemichannels in astrocytes was shown to impact synaptic activity and plasticity. The first evidence comes from hippocampal acute slices in which a decrease in extracellular Ca2+ concentration is generated by the local photolysis of a photolabile Ca2+ buffer. This opens Cx43 hemichannels in astrocytes through which ATP is released and activates P2Y1 purinergic receptors on a subset of inhibitory interneurons, initiating the generation of spikes by interneurons that in turn enhances synaptic inhibition at glutamatergic synapses. A similar neuroglial loop of interaction acting as a negative feedback mechanism is expected to occur during excessive glutamatergic activity that is associated with a decrease in extracellular Ca2+ concentration. The second set of evidence comes from in vivo experiments in which Cx43 hemichannels were inhibited. In this study, the rat basolateral amygdala was microinfused with the TAT-L2 peptide that selectively inhibits Cx43 hemichannels assumed to be only present in astrocytes. Such in vivo blockade of Cx43 hemichannels during memory consolidation induces amnesia for auditory fear conditioning, as assessed 24 h after training, without affecting short-term memory, locomotion, or shock reactivity. Moreover, the amnesic effect is transitory, specific for memory consolidation. Learning capacity recovers after co-infusion of the Cx43 hemichannel blocker and a mixture of gliotransmitters including glutamate and ATP. These observations suggest that gliotransmission mediated by Cx43 hemichannels is necessary for fear memory consolidation at the basolateral amygdala. Thus, the study of represents the first in vivo demonstration of a physiological role for astroglial Cx43 hemichannels in brain cognitive function. Finally, an in vivo contribution of Panx1 channels to cognition has recently been addressed by using a Panx1 knock out mouse. This constitutive knock out mouse was found to have increased excitability and potently enhanced early and persistent long-term potentiation (LTP) responses in the CA1 hippocampal region with additionally impaired spatial memory and object recognition memory. However, while the expression of Panx1 is well documented in hippocampal neurons, its detection at the membrane of astrocytes is still debated (; but see, ). Consequently, it is premature to state that these Panx1-dependent changes in hippocampal models of learning and memory have an astroglial origin.

34While there are now a number of reports indicating that the expression of glial Cxs changes in many brain pathologies and injuries, this remains to be investigated in detail for Panxs. On this basis, the question of Cx channels contributing to neuronal death and brain diseases has been debated for many years. Indeed, opposite perspectives regarding the roles of glial Cxs in neuronal death have been discussed and reviewed (see;;;;;; ). In general, this duality was attributed to differences in (i) the experimental models (in vitro, ex vivo, and in vivo) or the protocols used to induce neuronal death, (ii) the mode (pharmacology versus transgenic animals) of Cx channel inhibition selected, (iii) the type of neuronal death or pathology investigated, and (iv) the time points considered. However, retrospectively the opposite roles attributed to Cx channels could be simply due to the fact that for a long time this question was solely taking into account the gap junctional function of Cxs. Indeed, in most cases, interpretation of the data was not considering the hemichannel and channel functions of Cxs and Panxs, respectively. Nevertheless, the development of compounds and transgenic animals that discriminate between Cx gap junction channels, Cx hemichannels and Panx channels (see above) has allowed identification of their respective contribution in various in vitro and ex vivo models of brain pathologies (see;; ). Although hemichannels have been reported to be involved in several brain pathologies or injuries (reviewed in ) as well as infection, we will illustrate this situation by two recent examples that demonstrate the impact of glial Cx hemichannel and Panx channel activation in cells on neuronal death.

35Modifications in the pattern of Cx and Panx expression in glia are associated with phenotypic changes observed during neuroinflammation and the “reactive gliosis” that is a common feature of most brain diseases (see ). Indeed, activated microglia and reactive astrocytes exhibit changes in their respective pattern of expression for Panx1 and Cx43 as well as in their hemichannel function leading to neuronal damage and death. When an inflammatory context is mimicked in vitro by using LPS treatment, Cx43 hemichannel activity is triggered in astrocytes through the production of TNF-α and IL-1β by activated microglia. These events are not observed when astrocytes are cultured from Cx43 knock out mice. Furthermore, in neuron–astrocyte co-cultures the N-methyl-D-aspartate (NMDA)-induced neuronal excitotoxicity is reinforced by TNF-α + IL-1β treatment, while such a potentiating effect is not observed in cultures of neurons alone treated with the two cytokines. This observation suggests that the well-known protective role of astrocytes against excitotoxicity is impaired under pro-inflammatory conditions when astroglial Cx43 hemichannels are activated. Neuron–astrocyte co-cultures, made with astrocytes from Cx43 knock out and neurons from wild-type mice, revealed that the NMDA excitotoxicity is not reinforced by the pro-inflammatory treatment applied on these co-cultures made with Cx43-deficient astrocytes. This result suggests that astroglial Cx43 is involved in the potentiated neurotoxicity induced by pro-inflammatory treatments. Interestingly, application (<30 min) of mimetic peptides (Gap26, Gap27) that inhibit Cx43 hemichannels and have no effect on Cx43 gap junctional communication, prevents the potentiated neurotoxicity response induced by pro-inflammatory cytokines.

36Connexin hemichannels might also play a role in the pathological context of neurodegenerative diseases. Indeed, it is becoming increasingly clear that glial cells are critical players in the progressive neurodegeneration of Alzheimer’s disease. Among others, the amyloid cascade represents one of the key pathways involved in the pathogenesis of the disease (see ). Using separate primary cultures and co-cultures of microglia, astrocytes, and neurons it was shown that treatment with the β-amyloid peptide (Aβ) first activates microglia, resulting in the opening of Cx43 hemichannels and Panx1 channels in these cells. Such channel activations allow the release of glutamate and ATP that impair neuronal survival. Aβ-activated microglia also produces TNF-α and IL-1β that, as indicated above, induce Cx43 astroglial hemichannel activation that again results in the release of glutamate and ATP. In fine these gliotransmitters originating from glial cells trigger neuronal damage and induce their death. Thus, such a cascade of Cx glial hemichannel and Panx channel activation could partially account for the progression of the neurodegenerative disease. These in vitro and ex vivo observations could be related to the changes in Cx43 expression observed in reactive astrocytes that contact amyloid deposit in a murine model of Alzheimer’s disease and in brain from Alzheimer’s patients.

37During the last decade our view and understanding of the structure, mutations, properties, and biological roles of Cx channels has been incredibly enlarged, and to some extent this is also true for Panx channels. Interestingly, in glial cells this feature is exemplary because in addition to their well-documented molecular support for their two channel functions, non-channel functions have been also identified for Cxs, including cell adhesion mechanisms involved in radial migration of cortical neurons, modulation of P2Y purinergic receptors signaling, regulation of cytoskeletal dynamics and control of gene expression. Such a view could even be further enlarged by the recent report of another new class of membrane protein called calcium homeostasis modulator CALHM1 that shares structural similarities with Cx, Panx, and innexin channels and functional properties similar to hemichannels, such as ATP release in taste transduction.

38In the present review, we have focused on a Cx/Panx-based channel function which 10 years ago was only sparsely considered in the central nervous system and in neuroglial interactions. Since then, the identification of external and intracellular signals that trigger the activation of hemichannels, the understanding of their biophysics and their regulation have provided a better understanding of the roles that glial Cxs and Panxs play in healthy and diseased brains. Indeed, as reported above, there is now strong evidence to state that Cx and Panx channels in glia have an impact on neuronal activity and survival. However, there are at least two aims that remain to be achieved in order to determine whether and how the contribution of glial Cxs and/or Panxs is causal or secondary in normal and pathological situations. One is to distinguish what is due to gap junction channels versus hemichannels, the other is to discriminate between Cx hemichannels and Panx channels. To achieve these goals it is essential to have available pharmacological and genetic tools with glial cell type, Cx type, and Panx type specificities (see ). Through a simple pharmacological approach this is unlikely to be achieved since Cx and Panx channels are sensitive to the same agents so far described. However, strategies using short-term treatment with mimetic peptides or antibodies targeting extracellular domains could be developed for all the glial Cxs/Panxs and their specificity demonstrated. In contrast, due to the cytoplasmic location of their two channel ends, gap junction channels are likely less easy to target by a pharmacological strategy. Another more accurate approach consists in the development of transgenic mice with appropriate mutations that prevent channel pairing without affecting hemichannel activity or that block one but not the other channel function. Alternatively, small interfering RNA (siRNA) and transgenic/viral techniques have been used with some success (see ). But here again, the expression of multiple Cxs in glial cells makes the task difficult with genetic and molecular approaches. A recent report by Nedergaard’s group of an enhanced synaptic plasticity (LTP) due to the engraftment of human glial progenitors into immunodeficient mice gives weight to achieve such difficult challenges. Indeed, upon maturation in these grafted mice, human astrocytes were shown to be coupled to host astrocytes, suggesting a contribution of Cx channels in neuroglial interactions during synaptic plasticity. In line with such contribution, double Cx43/Cx30 knock out is associated with reduced LTP. Also, the observation of an up-regulation of astroglial Cxs in reactive astrocytes contacting amyloid plaques in a transgenic murine model of Alzheimer’s disease has been validated in human patient brain samples, giving another example of the value in searching for strategies that block Cx channels.

39The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.