S-Nitrosylation Inhibits Pannexin 1 Channel Function
| Autor: | Marie Billaud, Alexander W. Lohman, Joanna K. Sandilos, Adam C. Straub, Silvia Penuela, Norbert Leitinger, Janelle L. Weaver, Dale W. Laird, Douglas A. Bayliss, Brant E. Isakson, Rachael Griffiths |
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| Rok vydání: | 2012 |
| Předmět: |
Mutation
Missense Nerve Tissue Proteins Nitric Oxide Biochemistry Connexins Dithiothreitol Nitric oxide Mice chemistry.chemical_compound Adenosine Triphosphate Quinoxalines Membrane Biology Animals Humans Nitric Oxide Donors Molecular Biology Oxadiazoles HEK 293 cells Guanylate cyclase activity Wild type Cell Biology S-Nitrosylation Pannexin Nitro Compounds Glutathione Molecular biology Cell biology Quaternary Ammonium Compounds HEK293 Cells Amino Acid Substitution chemistry Mutation Oxidation-Reduction Adenosine triphosphate |
| Zdroj: | Journal of Biological Chemistry. 287:39602-39612 |
| ISSN: | 0021-9258 |
| Popis: | S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1(C40A), Panx1(C346A), and Panx1(C426A)). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1(C40A/C346A)) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release. |
| Databáze: | OpenAIRE |
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