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Complex interplay between Pannexin 1 cysteine residues and channel regulation
Author(s) -
Lohman Alexander W,
Weaver Janelle L,
Billaud Marie,
Bayliss Doug A,
Isakson Brant E
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1131.7
Subject(s) - cysteine , pannexin , hek 293 cells , transfection , s nitrosylation , chemistry , patch clamp , nitric oxide , alanine , microbiology and biotechnology , nitrosylation , mutant , biochemistry , biophysics , biology , amino acid , intracellular , gene , gap junction , enzyme , receptor , organic chemistry , connexin
Post‐translational modification of proteins by S‐nitrosylation at unique cysteine residues can profoundly affect protein function. We recently demonstrated the presence of pannexin 1 (Panx1) channels in the vasculature and thus investigated whether Panx1 can be S‐nitrosylated and if this modification affects channel activity. Application of S‐nitrosogluathione (GSNO), a nitric oxide (NO) donor, to HEK cells transfected with wild type (WT) Panx1 caused S‐nitrosylation of the protein as detected by the biotin switch assay. To identify which cysteine was being S‐nitrosylated, we performed multiple cysteine to alanine mutations (C40A, C66A, C346A, C426A) in the murine Panx1 gene. Mutation of cysteine 426 blocked S‐nitrosylation of Panx1, indicating this residue as the site of modification. To assess functional effects of Panx1 S‐nitrosylation, we subjected HEK cells transfected with WT or our Panx1 cysteine mutants to whole‐cell patch clamp recordings. Application of GSNO dramatically attenuated the Panx1 current in HEK cells expressing WT Panx1. However, C426A current was still inhibited similar to WT Panx1 current, whereas C40A and C346A Panx1 channel current were not altered by GSNO. Lastly, we measured dye uptake and ATP release to correlate electrophysiology data. This data suggests a complex interplay between cysteine regulation of pore function and NO.