Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107

Abstract

Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in the rapid release of Ca(2+) from the sarcoplasmic reticulum and muscle contraction. Dissociation of the small FK506 binding protein 12 subunit (FKBP12) increases RyR1 activity and impairs muscle function. The 1,4-benzothiazepine derivative JTV519, and the more specific derivative S107 (2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here, we report a high degree of nonspecific and specific low affinity [(3)H]S107 binding to SR vesicles. SR vesicles enriched in RyR1 bound ∼48 [(3)H]S107 per RyR1 tetramer with EC(50) ∼52 µM and Hillslope ∼2. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. S107 increased FKBP12 binding to RyR1 in SR vesicles in the presence of reduced glutathione and the NO-donor NOC12, with no effect in the presence of oxidized glutathione. Addition of 0.15 µM FKBP12 to SR vesicles prevented FKBP12 dissociation; however, in the presence of oxidized glutathione and NOC12, FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 µM myoplasmic FKBP12 and was attenuated by S107. In single channel measurements with FKBP12-depleted RyR1s, in the absence and presence of NOC12, S107 augmented the FKBP12-mediated decrease in channel activity. The data suggest that S107 can reverse the harmful effects of redox active species on SR Ca(2+) release in skeletal muscle by binding to RyR1 low affinity sites.

Figure 1. [³H]S107 binding to SR vesicles.

(A) Time course of total, nonspecific and specific [³H]S107 binding to SR vesicles incubated with 0.2 µM [³H]S107 in 0.25 M KCl, 20 mM imidazole, pH 7.0, 50 µM free Ca²⁺ and protease inhibitors for 1 to 9 h at 24°C. Nonspecific binding was determined by measuring [³H]S107 binding to SR vesicles heat-inactivated for 10 min at 95°C. Data are the mean ± SD of 3 experiments. (B) Total, nonspecific and specific [³H]S107 binding to SR vesicles incubated with 0.1 µM [³H]S107 and 0.1 to 100 µM S107 as above for 7 h at 24°C. Nonspecific binding was determined as in A. Specific binding curve was obtained using four parameter logistic equation shown in Materials and Methods.

Figure 2. Effects of FK506 and S107 on FKBP12 dissociation from SR vesicles in the presence of GSH and GSSG.

(A) Representative immunoblot of SR vesicles not treated and treated with FK506. SR vesicles were incubated with 10 µM FK506 as described in Materials and Methods, followed by centrifugation to remove FK506 and dissociated FKBP12. (B and C) FKBP12 dissociation from SR vesicles in the presence of GSH and GSSG. Immunoblots of SR vesicles not treated with FK506 were incubated for 1 and 20 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 50 µM free Ca²⁺, protease inhibitors, and the indicated concentrations and ratios (5 mM total glutathione) of GSH and GSSG in the absence and presence of 44 µM S107. Free FKBP12 was removed by centrifugation. Data were normalized to SR vesicles not incubated (gray bar, 0 min) and are the mean ± SEM of 4–5 experiments. *p<0.05 compared to SR vesicles at 0 min not treated with S107.

Figure 3. FKBP12 dissociation from SR vesicles in the presence of NOC12.

(A–C) SR vesicles not treated with FK506 were incubated for 5 h at 24°C with or without 0.10 mM NOC12 in the absence and presence of 44 µM S107 in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca²⁺ and protease inhibitors. S-nitrosylation was stopped by centrifugation. Resuspended samples were separated on 8–20% (FKBP12) and 3–12% (RyR1 and Cys-SNO) gradient SDS-PAGE gels and transferred to nitrocellulose membranes to detect S-nitrosylation of RyR1, and FKBP12 and RyR1 proteins. Data are the mean ± SEM of 4 determinations. *p<0.05 compared to control samples (B) and samples with NOC12 and S107 (C). (D and E) SR membranes were incubated with and without 44 µM S107 and 0.1 mM NOC12 at 24°C for 90 min, solubilized, and immunoprecipitated as described in Methods. Immunoblots of RyR1 and FKBP12 are shown. Data are the mean ± SEM of 4 experiments. ^*p<0.05 compared to control samples and samples incubated with NOC12 and S107.

Figure 4. Effects of NOC12 and S107 on [³H]ryanodine binding to RyR1.

(A) Dependence of [³H]ryanodine binding on NOC12 concentration. SR vesicles not treated with FK506 were incubated for 5 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca²⁺, protease inhibitors and the indicated concentrations of NOC12 in the presence (•) and absence (○) of 44 µM S107. Data are the mean ± SEM of 4 experiments. *p<0.05 compared to vesicles without S107. (B) Dependence of [³H]ryanodine binding to RyR1 on S107 concentration. SR vesicles were incubated as in A in the absence (○) and presence of 50 µM NOC12 (•) and the indicated concentrations of S107. Data are the mean ± SEM of 4 experiments. *p<0.05 compared to vesicles with 50 µM NOC12 and without S107. (C) Specific [³H]ryanodine binding to SR vesicles containing (−FK506) and depleted (+FK506) of FKBP12. [³H]Ryanodine binding was determined in the presence of 50 µM NOC12 and the absence and presence of 44 µM S107. Data are the mean ± SEM of 8 experiments. *p<0.05 compared to vesicles treated with FK506 and incubated in the absence of S107, ^#p<0.05 compared to vesicles not treated with FK506 and incubated in the absence of S107.

Figure 5. Effect of S107 on the stability of FKBP12-RyR1 complex in skeletal muscle homogenates.

(A and B) Skeletal muscle homogenates were incubated without (control) or with 5 mM GSH, 5 mM GSSG or 0.10 mM NOC12 in the absence or presence of 44 µM S107 for 20 h at 24°C. Unbound FKBP12 was removed by centrifugation and the amounts of RyR1 and FKBP12 were detected using anti-RyR1 and anti-FKBP12 antibodies. Homogenates incubated without glutathione and NOC12 served as control. Data are the mean ± SEM of 8 experiments. *p<0.05 compared to control homogenates without S107. ^#p<0.05 compared to homogenates incubated with NOC12 in the absence of S107.

Figure 6. Effects of S107 on FKBP12 binding to RyR1.

(A) Immunoblots of RyR1 and FKBP12. SR vesicles depleted of FKBP12 were incubated for 20 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca²⁺, protease inhibitors and 10 nM FKBP12 without or with 44 µM S107 in the absence and presence of 5 mM GSH, 5 mM GSSG or 0.10 mM NOC12. SR vesicles not treated with FK506 and incubated without glutathione served as control. (B) SR vesicles not treated with FK506 served as control. Data are the mean of 5–7 experiments. *p<0.05 compared to FKBP12-depleted vesicles incubated without S107 in the presence of GSH and NOC12, respectively, as determined by paired Student’s t-test.

Figure 7. Time course of FKBP12 binding in the presence and absence of S107.

(A) Immunoblots of RyR1 and FKBP12. SR vesicles treated with FK506 were incubated in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca²⁺, protease inhibitors and 1 µM FKBP12 for the indicated times in the presence of 5 mM GSH, and absence or presence of 44 µM S107. FKBP12 binding was stopped by centrifugation. (B) Data are the mean ± SEM of 7 experiments. They were corrected for amounts of FKBP12 associated with FK506 treated SR vesicles kept on ice and normalized to SR vesicles not treated with FK506 (gray bar). *p<0.05 compared to SR vesicles not treated with FK506 and not incubated with FKBP12 and S107. #p<0.05 compared to FKBP12-depleted SR vesicles incubated for the same time (10 min or 2 h) in the presence of FKBP12 but absence of S107.

Figure 8. Single channel measurements in the absence and presence of FKBP12 and S107.

(A) SR vesicles not treated (top trace) or treated with FK506 (traces 2 and 5) were incubated for 30 min at 24°C without addition (traces 1 and 2), with 25 µM S107 (trace 3), 5 µM FKBP12 (trace 4) or 25 µM S107 plus 5 µM FKBP12 (bottom trace) in 0.3 M sucrose, 0.25 M KCl, 20 mM imidazole, pH 7.0, and protease inhibitors. Vesicles were then fused to a lipid bilayer and recorded at 2 µM cis cytoplasmic Ca²⁺ and −35 mV as described in Materials and Methods. Representative single channel currents (downward deflections from closed levels, c–) (left) and current histograms (right) are shown. (B) Single channel data were obtained as described in A. Data are the mean ± SEM of 4–12 single channel recordings. ^*p<0.05 compared to RyR1 not treated with FK506 in the absence of S107 and FKBP12. ^#p<0.05 compared to FK506-treated RyR1 incubated in the absence of S107 and FKBP12. ^%p<0.05 compared to FK506-treated RyR1 incubated with FKBP12 in absence of S107. p values were determined by Student’s t-test.