title: S-nitrosylation inhibits pannexin 1 channel function.
abstract: 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.
authors.affiliation: Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Lohman
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Weaver
authors.forename: Marie
authors.initials: M
authors.lastname: Billaud
authors.forename: Joanna K
authors.initials: JK
authors.lastname: Sandilos
authors.forename: Rachael
authors.initials: R
authors.lastname: Griffiths
authors.forename: Adam C
authors.initials: AC
authors.lastname: Straub
authors.forename: Silvia
authors.initials: S
authors.lastname: Penuela
authors.forename: Norbert
authors.initials: N
authors.lastname: Leitinger
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Bayliss
authors.forename: Brant E
authors.initials: BE
authors.lastname: Isakson
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009574:Nitro Compounds / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology
chemical_list: C494582:1H-(1,2,3)oxadiazolo(4,4-a)quinoxalin-1-one; D017630:Connexins; D009419:Nerve Tissue Proteins; D020030:Nitric Oxide Donors; D009574:Nitro Compounds; D010069:Oxadiazoles; C492764:PANX1 protein, human; C503631:Panx1 protein, mouse; D000644:Quaternary Ammonium Compounds; D011810:Quinoxalines; C116027:S-nitroglutathione; C406802:diethylammonium-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D005978:Glutathione; D004229:Dithiothreitol