The mechanism of inhibition of ryanodine receptor channels by imperatoxin I, a heterodimeric protein from the scorpion Pandinus imperator.
We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.
Zamudio
F Z
FZ
Department of Molecular Recognition and Structural Biology, Biotechnology Institute, National Autonomous University of Mexico, Cuernavaca, Morelos 62271, Mexico.
Conde
R
R
Arévalo
C
C
Becerril
B
B
Martin
B M
BM
Valdivia
H H
HH
Possani
L D
LD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015723:Gene Library; D007700:Kinetics; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D012605:Scorpions; D017386:Sequence Homology, Amino Acid; D013552:Swine
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018076:DNA, Complementary; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D009124:Muscle Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012604:Scorpion Venoms; C105848:imperatoxin I; D012433:Ryanodine; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium