Astrocytes have a role in maintaining normal neuronal functions, some of which depend on connexins, protein subunits of gap junction channels and hemichannels. Under inflammatory conditions, microglia release cytokines, including interleukin-1β and tumor necrosis factor-α, that reduce intercellular communication via gap junctions. Now, we demonstrate that either conditioned medium harvested from activated microglia or a mixture of these two cytokines enhances the cellular exchange with the extracellular milieu via Cx43 hemichannels. These changes in membrane permeability were not detected in astrocytes cultured from Cx43 knock-out mice and were abrogated by connexin hemichannel blockers, including La³⁺, mimetic peptides, and niflumic acid. Both the reduction in gap junctional communication and the increase in membrane permeability were mediated by a p38 mitogen-activated protein kinase-dependent pathway. However, the increase in membrane permeability, but not the gap junction inhibition, was rapidly reversed by the sulfhydryl reducing agent dithiothreitol, indicating that final regulatory mechanisms are different. Treatment with proinflammatory cytokines reduced the total and cell surface Cx43 levels, suggesting that the increase in membrane permeability was attributable to an increase in hemichannels activity. Indeed, unitary events of ∼220 pS corresponding to Cx43 hemichannels were much more frequent in astrocytes treated with microglia conditioned medium than under control conditions. Finally, the effect of cytokines enhanced the uptake and reduced the intercellular diffusion of glucose, which might explain changes in the metabolic status of astrocytes under inflammatory conditions. Accordingly, this opposite regulation may affect glucose trafficking and certainly will modify the metabolic status of astrocytes involved in brain inflammation.

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. Antiox. Redox Signal. 11, 369–399.

Gap junction (GJ) channels assembled from connexin (Cx) proteins provide a structural basis for direct electrical and metabolic cell–cell communication. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we demonstrate that in response to transjunctional voltage (V _j ) Cx43/Cx45 heterotypic GJs exhibit both V _j -gating and dye transfer asymmetries. The later is affected by ionophoresis of charged fluorescent dyes and voltage-dependent gating. We demonstrate that small differences in resting (holding) potentials of communicating cells can fully block (at relative negativity on Cx45 side) or enhance (at relative positivity on Cx45 side) dye transfer. Similarly, series of high frequency V _j pulses resembling bursts of action potentials (APs) can fully block or increase the transjunctional flux (J _j ) of dye depending on whether pulses are generated in the cell expressing Cx43 or Cx45, respectively. Asymmetry of J _j -V _j dependence is enhanced or reduced when ionophoresis and V _j -gating act synergistically or antagonistically, whereas single channel permeability (P _γ ) remains unaffected. This modulation of intercellular signaling by V _j can play a crucial role in many aspects of intercellular communication in the adult, in embryonic development, and in tissue regeneration.

Neuronal gap junction (GJ) channels composed of connexin36 (Cx36) play an important role in neuronal synchronization and network dynamics. Here we show that Cx36-containing electrical synapses between inhibitory neurons of the thalamic reticular nucleus are bidirectionally modulated by changes in intracellular free magnesium concentration ([Mg²⁺]_i). Chimeragenesis demonstrates that the first extracellular loop of Cx36 contains a Mg²⁺-sensitive domain, and site-directed mutagenesis shows that the pore-lining residue D47 is critical in determining high Mg²⁺-sensitivity. Single-channel analysis of Mg²⁺-sensitive chimeras and mutants reveals that [Mg²⁺]_i controls the strength of electrical coupling mostly via gating mechanisms. In addition, asymmetric transjunctional [Mg²⁺]_i induces strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. We suggest that Mg²⁺-dependent synaptic plasticity of Cx36-containing electrical synapses could underlie neuronal circuit reconfiguration via changes in brain energy metabolism that affects neuronal levels of intracellular ATP and [Mg²⁺]_i.