Use-dependent inhibition of the skeletal muscle ryanodine receptor by the suramin analogue NF676

Abstract

The skeletal muscle Ca2+ release channel, the ryanodine receptor, is activated by the trypanocidal drug suramin via the calmodulin-binding site. As calmodulin activates and inhibits the ryanodine receptor depending on whether Ca2+ is absent or present, suramin analogues were screened for inhibition of the ryanodine receptor. Up to 300 microM, the novel suramin analogue, 4,4'-(carbonyl-bis(imino-4,1-phenylene-(2,5-benzimidazolylene)carbonylimino))-bis-benzenesulfonic acid disodium salt (NF676) was not able to significantly inhibit the basal [3H]ryanodine binding. However, kinetic analysis of the high affinity [3H]ryanodine binding elucidates a time-dependent increment of inhibition by NF676, which is indicative for an open channel blocker. Moreover, the ryanodine receptor was much more sensitive towards inhibition by NF676 when preactivated with caffeine or the nonhydrolysable ATP analogue, adenylyl-imidodiphosphate. Nonetheless, the suramin activated ryanodine receptor was not susceptible towards high-affinity NF676 inhibition, indicating an allosteric hindrance between the binding sites of suramin and NF676. In the line of this finding, NF676 per se was not capable to elute the purified ryanodine receptor from a calmodulin-Sepharose, but it prevented the elution by suramin. Other than suramin, NF676 did not inhibit the Ca2+ ATPase of the sarcoplasmic reticulum. However, suramin-induced Ca2+ release from sarcoplasmic reticulum was completely abrogated by preincubation with NF676. Taken together, we conclude from these data that NF676 represents a novel lead compound as a potent use-dependent blocker of the skeletal muscle ryanodine receptor via an allosteric interaction with the suramin-binding site.

Figure 1

Chemical structure of suramin and NF 676.

Figure 2

Inhibition of the RyR1 by NF676. (a) Sarcoplasmic reticulum membranes (75 μg) were incubated with 20 nM [³H]ryanodine for 45 min at 37°C in presence of NF676 at a calculated free Ca²⁺ concentration of 1.2 μM. (b) Kinetic analysis of the [³H]ryanodine binding in the absence and presence of 100 μM NF676 of five experiments. Asterisk represents statistical significance versus control (^*P=0.015; Students t-test). (c) [³H]ryanodine (20 nM) was allowed to associate to HSR membranes. After 120 min of incubation (time=0), the dissociation of [³H]ryanodine was triggered by the addition of 20 μM ryanodine in the absence or presence of 100 μM NF676. For comparison, the association was also allowed to proceed (control). The data represent mean±s.d.of a triplicate experiment, which was repeated twice with similar results.

Figure 3

Ca²⁺-dependent [³H]ryanodine binding in presence of NF676. Sarcoplasmic reticulum membranes (75 μg) were incubated with 20 nM [³H]ryanodine under conditions described in legend to Figure 2a. The free Ca²⁺ concentrations were adjusted by variation of the concentration of EGTA and CaCl₂. The incubation buffer containing only 1 mM EGTA but no supplemented CaCl₂ was set to be 1 nM free Ca²⁺. Incubations were carried out in the absence and presence of 100 μM NF676 or 20 μM ruthenium red. The data represent mean±s.e.m. of a triplicate experiment, which was repeated three times.

Figure 4

NF676 blocks the activated RyR1. (a) Under conditions described for Figure 2a, the basal [³H]ryanodine binding (control) was stimulated with 10 mM caffeine (caff.) and inhibited with the indicated concentrations of NF676 or ruthenium red (RR). (b–d) The [³H]ryanodine binding was inhibited with NF676 in absence or presence of 10 mM caffeine (b) or 0.5 mM AMP-PNP (c and d). Panel (b) and (d) depict the normalized [³H]ryanodine binding. The data represent mean±s.d. of a duplicate experiment, which was repeated two times.

Figure 5

Inhibition of suramin stimulated [³H]ryanodine binding by NF676. (a) Sarcoplasmic reticulum membranes (75 μg) were incubated under conditions given in legend to Figure 2a. The binding was carried out in the absence and presence of the indicated concentrations of NF676. (b) Inhibition of high-affinity [³H]ryanodine binding by NF676 in the absence or presence of 600 μM or 1 mM suramin. The data represent mean±s.d. of a duplicate experiment, which was repeated two times.

Figure 6

Modulation of the binding of calmodulin to the purified RyR1. (a–d) Elution of the purified RyR1 from a calmodulin-Sepharose matrix. The purified RyR1 was incubated with equilibrated calmodulin-Sepharose in presence of 200 μM free Ca²⁺ for 60 min at 4°C. The affinity matrix was washed three times and subsequently, RyR1 was eluted with buffer containing 10 μM calmodulin (a), 100 μM suramin (b), 1 mM NF676 (c) or 100 μM suramin plus 1mM NF676 (d). Residual protein trapped in the matrix is labelled ‘M'. Aliquots of input (lanes labelled ‘IN'), of last wash step (lanes labelled ‘W') and of the three implemented elution steps (lanes labelled ‘E1–E3'), were applied onto SDS–polyacrylamide gels. The proteins were visualized by silver staining and the high molecular mass range is depicted of the RyR1 indicated by ‘RyR'. The intensity of the RyR1 band was quantified by the Quanti Scan® software.

Figure 7

Effect of NF676 on Ca²⁺ uptake and Ca²⁺ release with HSR vesicles. (a–d) Ca²⁺ uptake and Ca²⁺ release were continuously monitored with a dual-wavelength photometer using 30 μM arsenazo III as a Ca²⁺-sensitive dye. Ca²⁺ uptake and suramin-induced Ca²⁺ release (a, c and d) were investigated in the absence (a) and presence of NF676 (b, c and d). The Ca²⁺ transients depict representative experiments (n=3–7).