The Role of the Microglial Sur1-Trpm4 Channel in the Calcium- and NFAT-dependent Expression of NOS2

Abstract

Background: Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca2+-sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca2+ influx, I hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as Nos2. Methods: I studied microglia in vivo and in primary culture from adult rats, and from wild type (WT), Abcc8-/- and Trpm4-/- mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS). To evaluate microglial responses, I used in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, quantitative real-time reverse-transcriptase PCR (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 ([Ca2+]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca2+]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca2+/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. Conclusion: Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca2+-sensitive gene expression, including Nos2.