Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine |
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Authors: | Michel Ronjat José Pablo Finkelstein Paola Llanos Luis Montecinos Hicham Bichraoui Michel De Waard Cecilia Hidalgo Ricardo Bull |
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Affiliation: | 1. Unité Inserm 836, Grenoble Institute of Neuroscience, Site Santé, BP 170, 38042 Grenoble, France;2. Université Joseph Fourier, Grenoble, France;3. Lab. Ex ICST, France;4. Centro de Estudios Moleculares de la Célula, Chile;5. Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile |
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Abstract: | The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca2+ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca2+ uptake completion prevented this response. Maurocalcine enhanced equilibrium [3H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg2+ inhibition of [3H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca2+] from 10 μM to 0.1 μM or at cytoplasmic [Mg2+] ≥ 30 μM. At 0.1 μM [Ca2+], only channels that displayed poor activation by Ca2+ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions. |
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Keywords: | Sulfhydryl oxidation Ryanodine binding Planar lipid bilayers Channel sub-conductance |
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