Neuronal excitotoxicity during stroke is caused by activation of unidentified large-conductance channels, leading to swelling and calcium dysregulation. We show that ischemic-like conditions [O(2)/glucose deprivation (OGD)] open hemichannels, or half gap junctions, in neurons. Hemichannel opening was indicated by a large linear current and flux across the membrane of small fluorescent molecules. Single-channel openings of hemichannels (530 picosiemens) were observed in OGD. Both the current and dye flux were blocked by inhibitors of hemichannels. Therefore, hemichannel opening contributes to the profound ionic dysregulation during stroke and may be a ubiquitous component of ischemic neuronal death.

Inflammatory bowel diseases (IBD) are chronic relapsing and remitting conditions associated with long-term gut dysfunction resulting from alterations to the enteric nervous system and a loss of enteric neurons1,2. The mechanisms underlying inflammation-induced enteric neuron death are unknown. Here we report using in vivo models of experimental colitis that inflammation causes enteric neuron death by activating a neuronal signaling complex comprised of P2X7 receptors (P2X7Rs), pannexin–1 (Panx1) channels, Asc and caspases. Inhibiting P2X7Rs, Panx1, Asc or caspase activity prevents inflammation-induced neuron cell death. Preservation of enteric neurons by inhibiting Panx1 in vivo prevented the onset of inflammation-induced colonic motor dysfunction. Panx1 expression is reduced in Crohn’s disease but not ulcerative colitis. We conclude that activation of neuronal Panx1 underlies neuron death and subsequent development of the abnormal gut motility in IBD. Targeting Panx1 represents a novel neuroprotective strategy to ameliorate the progression of IBD–associated dysmotility.

Pannexin-1 (Px1) is expressed at postsynaptic sites in pyramidal neurons, suggesting that these hemichannels contribute to dendritic signals associated with synaptic function. We found that, in pyramidal neurons, N-methyl-d-aspartate receptor (NMDAR) activation induced a secondary prolonged current and dye flux that were blocked with a specific inhibitory peptide against Px1 hemichannels; knockdown of Px1 by RNA interference blocked the current in cultured neurons. Enhancing endogenous NMDAR activation in brain slices by removing external magnesium ions (Mg2+) triggered epileptiform activity, which had decreased spike amplitude and prolonged interburst interval during application of the Px1 hemichannel blocking peptide. We conclude that Px1 hemichannel opening is triggered by NMDAR stimulation and can contribute to epileptiform seizure activity.

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.