Critical amino acid residues determine the binding affinity and the Ca2+ 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 Ca2+ 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.
Estève
Eric
E
INSERM EMI 9931, CEA, CIS, 17 Rue des Martyrs, 38054 Grenoble Cedex 09, France.
Smida-Rezgui
Sophia
S
Sarkozi
Sandor
S
Szegedi
Csaba
C
Regaya
Imed
I
Chen
Lili
L
Altafaj
Xavier
X
Rochat
Hervé
H
Allen
Paul
P
Pessah
Isaac N
IN
Marty
Isabelle
I
Sabatier
Jean-Marc
JM
Jona
Istvan
I
De Waard
Michel
M
Ronjat
Michel
M
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D012604:Scorpion Venoms / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D019837:Ryanodine Receptor Calcium Release Channel; D012604:Scorpion Venoms; C406406:maurocalcine; D012433:Ryanodine; D002118:Calcium