Kir6.1 Is the Principal Pore-Forming Subunit of Astrocyte but Not Neuronal Plasma Membrane K-ATP Channels

ATP-sensitive potassium channels (K-ATP channels) directly couple the energy state of a cell to its excitability, are activated by hypoxia, and have been suggested to protect neurons during disturbances of energy metabolism such as transient ischemic attacks or stroke. Molecular studies have demonstrated that functional K-ATP channels are octameric protein complexes, consisting of four sulfonylurea receptor proteins and four pore-forming subunits which are members of the Kir6 family of inwardly rectifying potassium channels. Here we show, using specific antibodies against the two known pore-forming subunits (Kir6.1 and Kir6.2) of K-ATP channels, that only Kir6.1 and not Kir6.2 subunits are expressed in astrocytes. In addition to a minority of neurons, Kir6.1 protein is present on hippocampal, cortical, and cerebellar astrocytes, tanycytes, and Bergmann glial cells. We also provide ultrastructural evidence that Kir6.1 immunoreactivity is primarily localized to distal perisynaptic and peridendritic astrocyte plasma membrane processes, and we confirm the presence of functional K-ATP channels in Bergmann glial cells by slice-patch-clamp experiments. The identification of Kir6.1 as the principal pore-forming subunit of plasma membrane K-ATP channels in astrocytes suggests that these glial K-ATP channels act in synergy with neuronal Kir6.2-mediated K-ATP channels during metabolic challenges in the brain.     

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.     

Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures

We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells.     

Potassium channel activity and glutamate uptake are impaired in astrocytes of seizure‐susceptible DBA/2 mice

Purpose: KCNJ10 encodes subunits of inward rectifying potassium (Kir) channel Kir4.1 found predominantly in glial cells within the brain. Genetic inactivation of these channels in glia impairs extracellular K⁺ and glutamate clearance and produces a seizure phenotype. In both mice and humans, polymorphisms and mutations in the KCNJ10 gene have been associated with seizure susceptibility. The purpose of the present study was to determine whether there are differences in Kir channel activity and potassium‐ and glutamate‐buffering capabilities between astrocytes from seizure resistant C57BL/6 (B6) and seizure susceptible DBA/2 (D2) mice that are consistent with an altered K⁺ channel activity as a result of genetic polymorphism of KCNJ10. Methods: Using cultured astrocytes and hippocampal brain slices together with whole‐cell patch‐clamp, we determined the electrophysiologic properties, particularly K⁺ conductances, of B6 and D2 mouse astrocytes. Using a colorimetric assay, we determined glutamate clearance capacity by B6 and D2 astrocytes. Results: Barium‐sensitive Kir currents elicited from B6 astrocytes are substantially larger than those elicited from D2 astrocytes. In addition, potassium and glutamate buffering by D2 cortical astrocytes is impaired, relative to buffering by B6 astrocytes. Discussion: In summary, the activity of Kir4.1 channels differs between seizure‐susceptible D2 and seizure‐resistant B6 mice. Reduced activity of Kir4.1 channels in astrocytes of D2 mice is associated with deficits in potassium and glutamate buffering. These deficits may, in part, explain the relatively low seizure threshold of D2 mice.     

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin-permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan-containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.     

Differential NMDA-dependent activation of glial cells in mouse hippocampus

In the hippocampus, the NMDA receptor is thought to be an important glutamate receptor involved in synaptic plasticity and in memory processes. Until recently, NMDA receptors have been considered solely as neuronal components, but some evidence suggests that glial cells in the hippocampus, and in particular astrocytes, also could be activated by NMDA applications. On the basis of their shape and electrophysiological properties (linear and rectified I/V curve), we describe two different populations of glial cells from GFAP-GFP transgenic mice that are activated differentially by NMDA. We found that linear glial cells were depolarized by NMDA that was not dependent on Ca2+ rise but partially involved a Ca2+ entry. Additionally, NMDA-induced depolarization of linear glial cells involved both a TTX-independent pathway likely through a direct activation, and a TTX-dependent pathway that required neuronal activity. The NMDA-induced depolarization in these cells was in part due to the activation of glutamate transporters and GABA B receptors. Furthermore, TTX-dependent NMDA-induced activation regulates the level of gap junction coupling between linear glial cells. In contrast, NMDA-induced depolarization in outward rectifying cells do not require a Ca2+ rise but are mediated directly by Ca2+ entry and are independent of glutamate transporters, GABA B and GABA A receptors. Our findings reveal that NMDA differentially activates hippocampal glial cells and the glial network through heterogeneous mechanisms in a cell-type specific manner.     

Down-regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.     

Kir potassium channel subunit expression in retinal glial cells: implications for spatial potassium buffering

To understand the role of different K(+) channel subtypes in glial cell-mediated spatial buffering of extracellular K(+), immunohistochemical localization of inwardly rectifying K(+) channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward-rectifying K(+) channel subunit Kir4.1 and for the strongly inward-rectifying K(+) channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell-mediated transport of extracellular K(+) away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K(+) currents leave the glial cells and enter extracellular "sinks," whereas K(+) influxes from neuronal "sources" into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the "cables" for spatial buffering currents may prevent an unwarranted outward leak of K(+), and, thus, avoid disturbances of neuronal information processing.     

Group I mGluR-Mediated Inhibition of Kir Channels Contributes to Retinal Muller Cell Gliosis in a Rat Chronic Ocular Hypertension Model

Müller cell gliosis, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), is a universal response in many retinal pathological conditions. Whether down-regulation of inward rectifying K(+) (Kir) channels, which commonly accompanies the enhanced GFAP expression, could contribute to Müller cell gliosis is poorly understood. We investigated changes of Kir currents, GFAP and Kir4.1 protein expression in Müller cells in a rat chronic ocular hypertension (COH) model, and explored the mechanisms underlying Müller cell gliosis. We show that Kir currents and Kir4.1 protein expression in Müller cells were reduced significantly, while GFAP expression was increased in COH rats, and these changes were eliminated by MPEP, a group I metabotropic glutamate receptors (mGluR I) subtype mGluR5 antagonist. In normal isolated Müller cells, the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppressed the Kir currents and the suppression was blocked by MPEP. The DHPG effect was mediated by the intracellular Ca(2+)-dependent PLC/IP(3)-ryanodine/PKC signaling pathway, but the cAMP-PKA pathway was not involved. Moreover, intravitreal injection of DHPG in normal rats induced changes in Müller cells, similar to those observed in COH rats. The DHPG-induced increase of GFAP expression in Müller cells was obstructed by Ba(2+), suggesting the involvement of Kir channels. We conclude that overactivation of mGluR5 by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir channels.     

Basic Science

Laura A. Jansen , Erik J. Uhlmann , Peter B. Crino , David H. Gutmann , and Michael Wong
Individuals with Tuberous Sclerosis Complex (TSC) frequently suffer from intractable epilepsy. To gain insight into the causes of epilepsy in TSC, we previously developed a mouse model of TSC with inactivation of the Tsc1 gene selectively in brain astrocytes (Tsc1GFAPCKO mice). Astrocytes, the main category of glial cells in brain, are important supporting cells in the brain, and also have direct effects on brain physiology, development, and repair. These mice develop progressive seizures, suggesting that astrocyte dysfunction may be involved in the development of epilepsy in TSC. Since one of the important functions of astrocytes is to limit sudden elevations of extracellular potassium in the brain, which would lead to neuronal excitability, in this study we investigated the hypothesis that impairment of potassium uptake may contribute to the development of seizures in Tsc1GFAPCKO mice. Astrocytes take up potassium from the extracellular space via a membrane channel, called the Kir channel. Cultured astrocytes from Tsc1GFAPCKO mice exhibited reduced Kir potassium currents and decreased expression of specific Kir channel protein subunits. mRNA expression of the same Kir subunits was also reduced in astrocytes from Tsc1GFAPCKO mice. Furthermore, we showed that the impairment in Kir channel function was reversed with drugs (roscovitine and retinoic acid) that modulate cell signaling pathways implicated in TSC. Lastly, hippocampal slices from Tsc1GFAPCKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced seizure-like activity. In conclusion, impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to increased neuronal excitability and the development of epilepsy in a mouse model of TSC.     

Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1-deficient astrocytes

PURPOSE: Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1(GFAP)CKO mice. METHODS: Kir channel function and expression were examined in cultured Tsc1-deficient astrocytes. Kir mRNA expression was analyzed in astrocytes microdissected from neocortical sections of Tsc1(GFAP)CKO mice. Physiological assays of astrocyte Kir currents and susceptibility to epileptiform activity induced by increased extracellular potassium were further studied in situ in hippocampal slices. RESULTS: Cultured Tsc1-deficient astrocytes exhibited reduced Kir currents and decreased expression of specific Kir channel protein subunits, Kir2.1 and Kir6.1. mRNA expression of the same Kir subunits also was reduced in astrocytes from neocortex of Tsc1(GFAP)CKO mice. By using pharmacologic modulators of signalling pathways implicated in TSC, we showed that the impairment in Kir channel function was not affected by rapamycin inhibition of the mTOR/S6K pathway, but was reversed by decreasing CDK2 activity with roscovitine or retinoic acid. Last, hippocampal slices from Tsc1(GFAP)CKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced epileptiform activity. CONCLUSIONS: Impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.     

Identification of an inward rectifier potassium channel gene expressed in mouse cortical astrocytes

These experiments identify an inward rectifier K+ (Kir) channel expressed in mouse cortical and white matter astrocytes at the molecular level. Messenger RNA for one of the known Kir channel genes, Kir4.1, is present at much higher levels in cortical astrocytes in primary culture than the other known Kir family members. In culture, the level of Kir4.1 mRNA is lower in proliferating cells and in cells cultured for 16 h under hypoxic conditions, compared to confluent cells. Partial differentiation of the astrocytes with dibutyryl cAMP or by coculture with neurons has no effect on the Kir4.1 mRNA level. In situ hybridization experiments show that Kir4.1 mRNA is broadly distributed in the adult brain, including the neocortex, the stratum pyrimadale of the hippocampus, and the piriform cortex. Immunostaining confirms that the Kir4.1 protein is expressed by cultured astrocytes and also by cocultured cortical neurons. Astrocytes and neurons display a patchy pattern of immunostaining, raising the possibility that the channels sort themselves in clusters in the plasma membrane. Stellate cells in the neocortex and white matter are immunoreactive for Kir4.1, and double immunofluorescence experiments show colocalization of Kir4.1 and glial acidic fibrillary protein (GFAP) on stellate cells in the white matter. The cloned mouse Kir4.1 cDNA, when expressed heterologously in HEK cells, gives rise to inactivating Kir channels similar to those recorded from cultured astrocytes. These results indicate that the Kir4.1 gene product forms a Kir channel, or is a subunit of the channel, in mouse cortical astrocytes both in culture and in vivo.     

Role of Kir4.1 channels in growth control of glia

The inwardly rectifying potassium channel Kir4.1 is widely expressed by astrocytes throughout the brain. Kir4.1 channels are absent in immature, proliferating glial cells. The progressive expression of Kir4.1 correlates with astrocyte differentiation and is characterized by the establishment of a negative membrane potential (> -70 mV) and an exit from the cell cycle. Despite some correlative evidence, a mechanistic interdependence between Kir4.1 expression, membrane hyperpolarization, and control of cell proliferation has not been demonstrated. To address this question, we used astrocyte-derived tumors (glioma) that lack functional Kir4.1 channels, and generated two glioma cell lines that stably express either AcGFP-tagged Kir4.1 channels or AcGFP vectors only. Kir4.1 expression confers the same K+ conductance to glioma membranes and a similar responsiveness to changes in [K+]o that characterizes differentiated astrocytes. Kir4.1 expression was sufficient to move the resting potential of gliomas from -50 to -80 mV. Importantly, Kir4.1 expression impaired cell growth by shifting a significant number of cells from the G2/M phase into the quiescent G0/G1 stage of the cell cycle. Furthermore, these effects could be nullified entirely if Kir4.1 channels were either pharmacologically inhibited by 100 microM BaCl2 or if cells were chronically depolarized by 20 mM KCl to the membrane voltage of growth competent glioma cells. These studies therefore demonstrate directly that Kir4.1 causes a membrane hyperpolarization that is sufficient to account for the growth attenuation, which in turn induces cell maturation characterized by a shift of the cells from G2/M into G0/G1.     

Complex rectification of Müller cell Kir currents

Although Kir4.1 channels are the major inwardly rectifying channels in glial cells and are widely accepted to support K+- and glutamate-uptake in the nervous system, the properties of Kir4.1 channels during vital changes of K+ and polyamines remain poorly understood. Therefore, the present study examined the voltage-dependence of K+ conductance with varying physiological and pathophysiological external [K+] and intrapipette spermine ([SP]) concentrations in Müller glial cells and in tsA201 cells expressing recombinant Kir4.1 channels. Two different types of [SP] block were characterized: "fast" and "slow." Fast block was steeply voltage-dependent, with only a low sensitivity to spermine and strong dependence on extracellular potassium concentration, [K+]o. Slow block had a strong voltage sensitivity that begins closer to resting membrane potential and was essentially [K+]o-independent, but with a higher spermine- and [K+]i-sensitivity. Using a modified Woodhull model and fitting i/V curves from whole cell recordings, we have calculated free [SP](in) in Müller glial cells as 0.81 +/- 0.24 mM. This is much higher than has been estimated previously in neurons. Biphasic block properties underlie a significantly varying extent of rectification with [K+] and [SP]. While confirming similar properties of glial Kir and recombinant Kir4.1, the results also suggest mechanisms underlying K+ buffering in glial cells: When [K+]o is rapidly increased, as would occur during neuronal excitation, "fast block" would be relieved, promoting potassium influx to glial cells. Increase in [K+]in would then lead to relief of "slow block," further promoting K+-influx.     

Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation

Evidence have accumulated that reverse glutamate uptake plays a key role in the pathophysiology of cerebral ischemia. Here, we investigated the effects of glial glutamate transporter dysfunction on neuronal survival using the substrate inhibitor of glutamate transporters, l-trans-pyrrolidine,2-4,dicarboxylate (PDC), that partly mimics reverse glutamate uptake. On mice primary cortical co-cultures of neurons and astrocytes, PDC treatment triggered an elevation of extracellular glutamate concentration, induced neuronal calcium influx and a massive NMDA receptor (NMDAR) mediated-neuronal death without having any direct agonist activity on NMDARs. We investigated the NMDAR subpopulation activated by PDC-induced glutamate release. PDC application led to the activation of both subtypes of NMDARs but the presence of astrocytes was required to activate NMDARs located extra-synaptically. Extrasynaptic NMDAR activation was also confirmed by the loss of neuronal mitochondrial membrane potential and the inhibition of pro-survival p-ERK signalling pathway. These data suggest that reverse glial glutamate uptake may trigger neuronal death through preferential activation of extrasynaptic NMDAR-related pathways.     

Pore-forming subunits of K-ATP channels, Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution in the rat brain

K-ATP channels consist of two structurally different subunits: a pore-forming subunit of the Kir6.0-family (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2, SUR2A, SUR2B) with regulatory activity. The functional diversity of K-ATP channels in brain is broad and of fundamental importance for neuronal activity. Here, using immunocytochemistry with monospecific antibodies against the Kir6.1 and Kir6.2 subunits, we analyze the regional and cellular distribution of both proteins in the adult rat brain. We find Kir6.2 to be widely expressed in all brain regions, suggesting that the Kir6.2 subunit forms the pore of the K-ATP channels in most neurons, presumably protecting the cells during cellular stress conditions such as hypoglycemia or ischemia. Especially in hypothalamic nuclei, in particular the ventromedial and arcuate nucleus, neurons display Kir6.2 immunoreactivity only, suggesting that Kir6.2 is the pore-forming subunit of the K-ATP channels in the glucose-responsive neurons of the hypothalamus. In contrast, Kir6.1-like immunolabeling is restricted to astrocytes (Thomzig et al. [2001] Mol Cell Neurosci 18:671-690) in most areas of the rat brain and very weak or absent in neurons. Only in distinct nuclei or neuronal subpopulations is a moderate or even strong Kir6.1 staining detected. The biological functions of these K-ATP channels still need to be elucidated.     

Glial hemichannels and their involvement in aging and neurodegenerative diseases

During the last two decades, it became increasingly evident that glial cells accomplish a more important role in brain function than previously thought. Glial cells express pannexins and connexins, which are member subunits of two protein families that form membrane channels termed hemichannels. These channels communicate intra- and extracellular compartments and allow the release of autocrine/paracrine signaling molecules [e.g., adenosine triphosphate (ATP), glutamate, nicotinamide adenine dinucleotide, and prostaglandin E2] to the extracellular milieu, as well as the uptake of small molecules (e.g., glucose). An increasing body of evidence has situated glial hemichannels as potential regulators of the beginning and maintenance of homeostatic imbalances observed in diverse brain diseases. Here, we review and discuss the current evidence about the possible role of glial hemichannels on neurodegenerative diseases. A subthreshold pathological threatening condition leads to microglial activation, which keeps active defense and restores the normal function of the central nervous system. However, if the stimulus is deleterious, microglial cells and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins, and ATP), which increase the activity of glial hemichannels, reducing the astroglial neuroprotective functions, and further reducing neuronal viability. Because ATP and glutamate are released via glial hemichannels in neurodegenerative conditions, it is expected that they contribute to neurotoxicity. More importantly, toxic molecules released via glial hemichannels could increase the Ca2+ entry in neurons also via neuronal hemichannels, leading to neuronal death. Therefore, blockade of hemichannels expressed by glial cells and/or neurons during neuroinflammation might prevent neurodegeneration.     

Glutamate release from astrocyte cell-line GL261 via alterations in the intracellular ion environment

Astrocytes modify and maintain neural activity and functions via gliotransmitter release such as, glutamate. They also change their properties and functions in response to alterations of ion environment resulting from neurotransmission; however, the direct evidence for whether intracellular ion alteration in astrocytes triggers gliotransmitter release is not indicated. Recent studies have reported that channelrhodopsin-2 (ChR2) is useful for alteration of intracellular ion environment in several types of cells with blue light exposure. Here, we show that ChR2-expressing GL261 (GLChR2) cells, clonal astrocytes, change their properties by photo-activation. Increased intracellular sodium and calcium ion concentrations and an altered membrane potential were observed in GLChR2 cells with blue light exposure. Alterations in the intracellular ion environment caused intracellular acidification and the inhibition of proliferation. In addition, it triggered glutamate release from GLChR2 cells. Glutamate from GLChR2 cells acted on N18 cells, clonal neuronal cells, as both a transmitter and neurotoxin depending on photo-activation. Our results show that the properties of ChR2-expressing astrocytes can be controlled by blue light exposure, and cation influx through photo-activated ChR2 might trigger functional cation influx via endogenous channels and result in the increase of glutamate release. Further, our results suggest that ChR2-expressing glial cells could become a useful tool in understanding the roles of glial cell activation and neural communication in the regulation of brain functions.