title: The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca(2+)-sensitive gene expression, including Nos2.
authors.affiliation: Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD, 21201-1595, USA. kurland.davidb@gmail.com.|Neurosurgery Research Laboratories, 10 S. Pine St, Baltimore, MD, 21201-1595, USA. kurland.davidb@gmail.com.
authors.forename: David B
authors.initials: DB
authors.lastname: Kurland
authors.affiliation: Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD, 21201-1595, USA.
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Gerzanich
authors.affiliation: Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD, 21201-1595, USA.
authors.forename: Jason K
authors.initials: JK
authors.lastname: Karimy
authors.affiliation: Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD, 21201-1595, USA.
authors.forename: Seung Kyoon
authors.initials: SK
authors.lastname: Woo
authors.affiliation: Department Cell Molecular Medicine, Laboratory Ion Channel Research, Campus Gasthuisberg, Herestraat 49-Bus 802, Leuven, 3000, Belgium.
authors.forename: Rudi
authors.initials: R
authors.lastname: Vennekens
authors.affiliation: Pharmakologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 366, Heidelberg, 69120, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Freichel
authors.affiliation: Department Cell Molecular Medicine, Laboratory Ion Channel Research, Campus Gasthuisberg, Herestraat 49-Bus 802, Leuven, 3000, Belgium.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nilius
authors.affiliation: Pacific Northwest Diabetes Research Institute, 720 Broadway, Seattle, WA, 98122, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bryan
authors.affiliation: Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD, 21201-1595, USA. msimard@smail.umaryland.edu.|Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA. msimard@smail.umaryland.edu.|Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA. msimard@smail.umaryland.edu.
authors.forename: J Marc
authors.initials: JM
authors.lastname: Simard
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003981:Diazoxide / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D064233:Sulfonylurea Receptors / Q000502:physiology*; D050053:TRPM Cation Channels / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*
chemical_list: C577508:Abcc8 protein, mouse; D064233:Sulfonylurea Receptors; D050053:TRPM Cation Channels; C476766:TRPM4 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D003981:Diazoxide
keywords: KATP; Microglia; NO; NOS2; Sur1-Trpm4; TLR4