Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine

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Abstract

We purified and characterized ryanotoxin, an ~11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduced unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 μM ryanotoxin was 163 ± 12 pS, that of the state induced by 1 μM ryanodine was 173 ± 26 pS, and that of control channels was 2.3-fold larger (396 ± 25 pS). The distribution of substate events induced by 1 μM RyTx was biexponential and was fitted with time constants ~10 times shorter than those fitted to the distribution of substates induced by 1 μM ryanodine. Bath-applied 5 μM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 μM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 μM without altering the maximum number (B(max)) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.

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Morrissette, J., Beurg, M., Sukhareva, M., & Coronado, R. (1996). Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine. Biophysical Journal, 71(2), 707–721. https://doi.org/10.1016/S0006-3495(96)79270-6

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