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Background  Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca 2+ -sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca 2+  influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as  Nos2 .    Methods  We studied microglia in vivo and in primary culture from adult rats, and from wild type,  Abcc8 −/− and  Trpm4 −/− mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation.    Results  In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of  Nos2  mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of  Abcc8  or  Trpm4  reduced  Nos2  upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca 2+ ] i ), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca 2+ ] i  induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca 2+ /calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation.    Conclusions  Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca 2+ -sensitive gene expression, including  Nos2 .

The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia