TY - JOUR
T1 - The structure of the C-terminal helical bundle in glutathione transferase M2-2 determines its ability to inhibit the cardiac ryanodine receptor
AU - Hewawasam, Ruwani
AU - Liu, Dan
AU - Casarotto, Marco G.
AU - Dulhunty, Angela F.
AU - Board, Philip G.
PY - 2010/8
Y1 - 2010/8
N2 - Ca2+ 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 Ca2+ 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 Ca2+ 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 2mM ATP and 10μM Ca2+ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40mV, which may develop during Ca2+ efflux, but not at -40mV. 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 Ca2+ release during diastole, which can lead to fatal arrhythmia.
AB - Ca2+ 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 Ca2+ 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 Ca2+ 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 2mM ATP and 10μM Ca2+ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40mV, which may develop during Ca2+ efflux, but not at -40mV. 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 Ca2+ release during diastole, which can lead to fatal arrhythmia.
KW - Calcium release from cardiac sarcoplasmic reticulum
KW - Calcium release from skeletal sarcoplasmic reticulum
KW - Cardiac RyR2 channels
KW - Glutathione transferase GSTM2-2
KW - Lipid bilayer single channel experiments
KW - Skeletal RyR1 channels
UR - http://www.scopus.com/inward/record.url?scp=77953356388&partnerID=8YFLogxK
U2 - 10.1016/j.bcp.2010.04.019
DO - 10.1016/j.bcp.2010.04.019
M3 - Article
SN - 0006-2952
VL - 80
SP - 381
EP - 388
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 3
ER -