Critical amino acid residues determine the binding affinity and the Ca ²⁺ release efficacy of maurocalcine in skeletal muscle cells

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [³H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC₅₀ of 12 nM. MCa decreases the sensitivity of [³H]ryanodine binding to inhibitory high Ca²⁺ concentrations and increases it to the stimulatory low Ca²⁺ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca ²⁺ efflux as demonstrated by MCa effects on Ca²⁺ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca²⁺ increase due to Ca²⁺ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg²⁴ for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [³H]ryanodine binding although with a decreased EC₅₀ and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca²⁺ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca²⁺ release from SR.