title: The inhibitory glutathione transferase M2-2 binding site is located in divergent region 3 of the cardiac ryanodine receptor.
abstract: The muscle-specific glutathione transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca(2+) release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855-1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca(2+) and improve contractility.
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australia.
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.forename: Ruwani
authors.initials: R
authors.lastname: Hewawasam
authors.forename: Yamuna
authors.initials: Y
authors.lastname: Karunasekara
authors.forename: Marco G
authors.initials: MG
authors.lastname: Casarotto
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020013:Calcium Signaling; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D014364:Tryptophan; D005982:Glutathione Transferase; C412602:glutathione S-transferase Mu 2