Background: Sur1-NCCa-ATP channels implicated in acute CNS injury are hypothesized to be formed by co-association of Sur1 and a nonselective cation channel.Results: Sur1 and Trpm4 form heteromers that exhibit pharmacological properties of Sur1 and biophysical properties of Trpm4.Conclusion: Sur1 and Trpm4 co-assemble to form the unique Sur1-Trpm4 channel.Significance: Identification of Sur1-Trpm4 channels has broad implications in acute CNS injuries.

Background:Most investigators of brain–computer interface (BCI) research believe that BCI can be achieved through induced neuronal activity from the cortex, but not by evoked neuronal activity. Motor imagery (MI)–based BCI is one of the standard concepts of BCI, in that the user can generate induced activity by imagining motor movements. However, variations in performance over sessions and subjects are too severe to overcome easily; therefore, a basic understanding and investigation of BCI performance variation is necessary to find critical evidence of performance variation. Here we present not only EEG datasets for MI BCI from 52 subjects, but also the results of a psychological and physiological questionnaire, EMG datasets, the locations of 3D EEG electrodes, and EEGs for non-task-related states.Findings:We validated our EEG datasets by using the percentage of bad trials, event-related desynchronization/synchronization (ERD/ERS) analysis, and classification analysis. After conventional rejection of bad trials, we showed contralateral ERD and ipsilateral ERS in the somatosensory area, which are well-known patterns of MI. Finally, we showed that 73.08% of datasets (38 subjects) included reasonably discriminative information.Conclusions:Our EEG datasets included the information necessary to determine statistical significance; they consisted of well-discriminated datasets (38 subjects) and less-discriminative datasets. These may provide researchers with opportunities to investigate human factors related to MI BCI performance variation, and may also achieve subject-to-subject transfer by using metadata, including a questionnaire, EEG coordinates, and EEGs for non-task-related states.

Astrocyte swelling occurs after central nervous system injury and contributes to brain swelling, which can increase mortality. Mechanisms proffered to explain astrocyte swelling emphasize the importance of either aquaporin-4 (AQP4), an astrocyte water channel, or of Na 1 -permeable channels, which mediate cellular osmolyte influx. However, the spatio-temporal functional interactions between AQP4 and Na 1 -permeable channels that drive swelling are poorly understood. We hypothesized that astrocyte swelling after injury is linked to an interaction between AQP4 and Na 1 -permeable channels that are newly upregulated. Here, using co-immunoprecipitation and F€ orster resonance energy transfer, we report that AQP4 physically co-assembles with the sulfonylurea receptor 1-transient receptor potential melastatin 4 (SUR1-TRPM4) monovalent cation channel to form a novel heteromultimeric water/ion channel complex. In vitro cell-swelling studies using calcein fluorescence imaging of COS-7 cells expressing various combinations of AQP4, SUR1, and TRPM4 showed that the full tripartite complex, comprised of SUR1-TRPM4-AQP4, was required for fast, high-capacity transmembrane water transport that drives cell swelling, with these findings corroborated in cultured primary astrocytes. In a murine model of brain edema involving cold-injury to the cerebellum, we found that astrocytes newly upregulate SUR1-TRPM4, that AQP4 co-associates with SUR1-TRPM4, and that genetic inactivation of the solute pore of the SUR1-TRPM4-AQP4 complex blocked in vivo astrocyte swelling measured by diolistic labeling, thereby corroborating our in vitro functional studies. Together, these findings demonstrate a novel molecular mechanism involving the SUR1-TRPM4-AQP4 complex to account for bulk water influx during astrocyte swelling. These findings have broad implications for the understanding and treatment of AQP4-mediated pathological conditions. K E Y W O R D Sastrocyte, ion channels, ions, osmosis, water

Background and Purpose Subarachnoid hemorrhage (SAH) can leave patients with memory impairments that may not recover fully. Molecular mechanisms are poorly understood, and no treatment is available. The sulfonylurea receptor 1–transient receptor potential melastatin 4 (Sur1-Trpm4) channel plays an important role in acute central nervous system injury. We evaluated upregulation of Sur1-Trpm4 in humans with SAH and, in rat models of SAH, we examined Sur1- Trpm4 upregulation, its role in barrier dysfunction and neuroinflammation, and its consequences on spatial learning. Methods We used Förster resonance energy transfer to detect coassociated Sur1 and Trpm4 in human autopsy brains with SAH. We studied rat models of SAH involving filament puncture of the internal carotid artery or injection of blood into the subarachnoid space of the entorhinal cortex. In rats, we used Förster resonance energy transfer and coimmunoprecipitation to detect coassociated Sur1 and Trpm4, we measured immunoglobulin G extravasation and tumor necrosis α overexpression as measures of barrier dysfunction and neuroinflammation, and we assessed spatial learning and memory on days 7 to 19. Results Sur1-Trpm4 channels were upregulated in humans and rats with SAH. In rats, inhibiting Sur1 using antisense or the selective Sur1 inhibitor glibenclamide reduced SAH-induced immunoglobulin G extravasation and tumor necrosis α overexpression. In models with entorhinal SAH, rats treated with glibenclamide for 7 days after SAH exhibited better platform search strategies and better performance on incremental and rapid spatial learning than vehicle-treated controls. Conclusions Sur1-Trpm4 channels are upregulated in humans and rats with SAH. Channel inhibition with glibenclamide may reduce neuroinflammation and the severity of cognitive deficits after SAH.

The engagement of integrin ␣7 in E63 skeletal muscle cells by laminin or anti-␣7 antibodies triggered transient elevations in the intracellular free Ca 2ϩ concentration that resulted from both inositol triphosphate-evoked Ca 2ϩ release from intracellular stores and extracellular Ca 2ϩ influx through voltage-gated, L-type Ca 2ϩ channels. The extracellular domain of integrin ␣7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin ␣7 in a manner highly dependent on the cytosolic Ca 2ϩ concentration. It appeared that intracellular Ca 2ϩ release was a prerequisite for Ca 2ϩ influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca 2ϩ release and Ca 2ϩ influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca 2ϩ channels on the cell surface.

BackgroundIn experimental autoimmune encephalomyelitis (EAE), deletion of transient receptor potential melastatin 4 (Trpm4) and administration of glibenclamide were found to ameliorate disease progression, prompting speculation that glibenclamide acts by directly inhibiting Trpm4. We hypothesized that in EAE, Trpm4 upregulation is accompanied by upregulation of sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which are highly sensitive to glibenclamide, and that Sur1-Trpm4 channels are required for EAE progression.MethodsEAE was induced in wild-type (WT) and Abcc8−/− mice using myelin oligodendrocyte glycoprotein 35–55 (MOG35–55). Lumbar spinal cords were examined by immunohistochemistry, immuno-Förster resonance energy transfer (immunoFRET), and co-immunoprecipitation for Sur1-Trpm4. WT/EAE mice were administered with the Sur1 inhibitor, glibenclamide, beginning on post-induction day 10. Mice were evaluated for clinical function, inflammatory cells and cytokines, axonal preservation, and white matter damage.ResultsSur1-Trpm4 channels were upregulated in EAE, predominantly in astrocytes. The clinical course and severity of EAE were significantly ameliorated in glibenclamide-treated WT/EAE and in Abcc8−/−/EAE mice. At 30 days, the lumbar spinal cords of glibenclamide-treated WT/EAE and Abcc8−/−/EAE mice showed significantly fewer invading immune cells, including leukocytes (CD45), T cells (CD3), B cells (CD20) and macrophages/microglia (CD11b), and fewer cells expressing pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17). In both glibenclamide-treated WT/EAE and Abcc8−/−/EAE mice, the reduced inflammatory burden correlated with better preservation of myelin, better preservation of axons, and more numerous mature and precursor oligodendrocytes.ConclusionsSur-Trpm4 channels are newly upregulated in EAE and may represent a novel target for disease-modifying therapy in multiple sclerosis.

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH), and may be responsible for important complications of SAH. Signaling by Toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NFκB) in microglia plays a critical role in neuronal damage after SAH. Three molecules derived from erythrocyte breakdown have been postulated to be endogenous TLR4 ligands: methemoglobin (metHgb), heme and hemin. However, poor water solubility of heme and hemin, and lipopolysaccharide (LPS) contamination have confounded our understanding of these molecules as endogenous TLR4 ligands. We used a 5-step process to obtain highly purified LPS-free metHgb, as confirmed by Fourier Transform Ion Cyclotron Resonance mass spectrometry and by the Limulus amebocyte lysate assay. Using this preparation, we show that metHgb is a TLR4 ligand at physiologically relevant concentrations. metHgb caused time- and dose-dependent secretion of the proinflammatory cytokine, tumor necrosis factor α (TNFα), from microglial and macrophage cell lines, with secretion inhibited by siRNA directed against TLR4, by the TLR4-specific inhibitors, Rs-LPS and TAK-242, and by anti-CD14 antibodies. Injection of purified LPS-free metHgb into the rat subarachnoid space induced microglial activation and TNFα upregulation. Together, our findings support the hypothesis that, following SAH, metHgb in the subarachnoid space can promote widespread TLR4-mediated neuroinflammation.