The structure of the C-terminal helical bundle in glutathione transferase M2-2 determines its ability to inhibit the cardiac ryanodine receptor Ruwani Hewawasam, Dan Liu, Marco G. Casarotto, Angela F. Dulhunty 1 , Philip G. Board 1, * Structural Biology Program, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia 1. Introduction The ryanodine receptor (RyR) functions as an ion channel that releases Ca 2+ from the sarcoplasmic reticulum (SR) and is essential for excitation contraction coupling and contraction in striated muscle. In previous studies we have shown that the human muscle specific glutathione transferase M2-2 (GSTM2-2) is a high affinity inhibitor of cardiac muscle ryanodine receptors (RyR2) and a weak activator of skeletal muscle ryanodine receptors (RyR1) [1,2]. Investigations of the function of endogenous modulators of RyR2 are important for understanding their contribution to cardiac output. In addition, inhibition of RyR2 is a potential strategy for the treatment of heart failure. The role of GSTM2-2 as an endogenous inhibitor of RyR2 is particularly important in the context of it helping to maintain low RyR2 activity during diastole. The cardiac RyR2 has few endogenous inhibitors and, unlike the skeletal RyR1, it is not sensitive to block by normal cytoplasmic concentrations of Mg 2+ of around 1 mM [3,4]. It is essential that RyR2 activity is low during diastole so that Ca 2+ concentrations are maintained at appropriate levels in the sarcoplasmic reticulum Ca 2+ stores. It is well documented that excessively active RyR2 channels are partly responsible for low store Ca 2+ levels and defective Ca 2+ release in heart failure. Stress-induced ventricular arrhythmias linked to mutations in RyR2 (or its associated proteins) that cause the channels that remain active during diastole, can cause sudden death in apparently healthy young individuals [5]. We have explored the regions of GSTM2-2 that interact with RyR2 and inhibit the channel in the hope of finding a region that might be developed to reduce the activity of overly active RyR2 channels in heart failure and stress-induced arrhythmias. Cross linking studies have shown that GSTM2-2 binds to RyR2 through its C-terminal domain (GSTM2-C) and that this a-helical domain can independently inhibit RyR2 function but has no effect on skeletal muscle RyR1 Ca 2+ channels [6]. Further dissection of GSTM2-C into a number of smaller peptides revealed that helix 6 is a core element that was essential for RyR2 inhibition. Previous studies [7] have shown that helix 6 plays a key role in global folding of the cytosolic GST family. However, we also found that a helix 6 peptide did not form an a-helix and was not an effective RyR2 inhibitor [6]. We have now made specific mutations in helix 6 within GSTM2-C to probe the relationship between helix 6 and the structure of GSTM2-C and its capacity to inhibit RyR2. The results provide further evidence for the importance of the stability Biochemical Pharmacology 80 (2010) 381–388 ARTICLE INFO Article history: Received 19 January 2010 Accepted 15 April 2010 Keywords: Glutathione transferase GSTM2-2 Cardiac RyR2 channels Skeletal RyR1 channels Calcium release from cardiac sarcoplasmic reticulum Calcium release from skeletal sarcoplasmic reticulum Lipid bilayer single channel experiments ABSTRACT Ca 2+ release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of glutathione transferase M2-2 (GSTM2- C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca 2+ release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca 2+ release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2 mM ATP and 10 mM Ca 2+ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40 mV, which may develop during Ca 2+ efflux, but not at 40 mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca 2+ release during diastole, which can lead to fatal arrhythmia. ß 2010 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 ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2010.04.019