The inhibitory glutathione transferase M2-2 binding site is located in divergent region 3 of the cardiac ryanodine receptor Dan Liu, Ruwani Hewawasam, Yamuna Karunasekara, Marco G. Casarotto, Angela F. Dulhunty 1 , Philip G. Board 1, * John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia 1. Introduction The ryanodine receptor (RyR) is an intracellular Ca 2+ ion channel that is essential for the process of excitation–contraction (E–C coupling) in the heart and skeletal muscle. There are three isforms of the RyR protein in mammals: with RyR1 preferentially expressed in skeletal muscle, RyR2 in cardiac muscle, and RyR3 in smooth muscle, neurones and other cell types. The identity of amino acid sequences between the three RyRs is 65–70%, with three regions, named the D1, D2, and D3 regions [1,2], that are dramatically divergent between each of the RyR proteins. The D1 region is located between residues 4254–4631 in RyR1 and between residues 4210–4562 in RyR2. The D2 region lies between residues 1342–1403 in RyR1 and between residues 1353–1397 in RyR2. Of particular interest in this study, the D3 region is located in a cytosolic domain found between residues 1872–1923 of RyR1 and between residues 1855–1890 of RyR2. There are many endogenous proteins and compounds that bind to RyRs and regulate their activity, but few are isoform-specific. Although it has been suggested that the divergent regions are responsible for the different isoform-specific properties of the mammalian RyRs, few isoform-specific properties have been experimentally attributed to a particular divergent region. Such allocation is clearly important as understanding the mechanisms that modulate the isoform-specific functions of RyR Ca 2+ channels could lead to the development of new therapeutic drugs to specifically enhance cardiac or skeletal muscle function. The glutathione transferases (GSTs) are a major family of phase II detoxification enzymes that conjugate the tripeptide glutathione (GSH) to a wide range of endogenous and exogenous toxins [3,4]. The structure of members of the GST superfamily is highly conserved and consists of an N-terminal domain with a characteristic thioredoxin fold and a strongly helical C-terminal domain. The active site and the GSH binding site are located on the N-terminal domain and residues involved in binding the hydro- phobic substrate are generally located in the C-terminal domain [5]. In recent years several members of the GST structural family have been implicated in other cellular processes including the catabolism of tyrosine [6], the synthesis of steroid hormones [7] and the regulation of stress activated kinases such as Jun N- terminal kinase [8] and ASK1 kinase [9,10]. In addition, members of the cytosolic GST structural family such as CLIC-2, GSTO1-1 and GSTM2-2 have been identified as a new class of endogenous RyR modulators [11]. These proteins have strong inhibitory actions on cardiac RyR2 channels, while the GST isoforms have weak excitatory effects on the skeletal RyR1 channels [11–15]. These results suggested that GSTs bind at different sites on RyR1 and Biochemical Pharmacology 83 (2012) 1523–1529 A R T I C L E I N F O Article history: Received 15 November 2011 Received in revised form 19 February 2012 Accepted 21 February 2012 Available online 1 March 2012 Keywords: Glutathione transferase GSTM2-2 Cardiac RyR2 channels Divergent region 3 of RyRs Skeletal RyR1 channels Inhibition of RyR2 channels Yeast two hybrid analysis A B S T R A C T 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. ß 2012 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +61 2 6125 4714; fax: +61 2 6125 4712. E-mail address: Philip.Board@anu.edu.au (P.G. Board). 1 Board and Dulhunty made equal senior author contributions to the manuscript. Contents lists available at SciVerse ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2012.02.020