Effects of recombinant imperatoxin A (IpTxa) mutants on the rabbit ryanodine receptor.
Imperatoxin A (IpTxa), a 3.7 kDa peptide from the African scorpion Pandinus imperator, is an agonist of the skeletal muscle ryanodine receptor (RyR1). In order to study the structure of the toxin and its effect on RyR1, IpTxa cDNA was PCR-amplified using 3 pairs of primers, and the toxin was expressed in E. coli. The toxin was further purified by chromatography, and various point mutants in which basic amino acids were substituted by alanine were prepared by site-directed mutagenesis. Studies of single channel properties by the planar lipid bilayer method showed that the recombinant IpTxa was identical to the synthetic IpTxa with respect to high-performance liquid chromatography mobility, amino acid composition and specific effects on RyR1. Mutations of certain basic amino acids (Lys19, Arg23, and Arg33) dramatically reduced the capacity of the peptide to activate RyRs. A subconductance state predominated when Lys8 was substituted with alanine. These results suggest that some basic amino acid residues in IpTxa are important for activation of RyR1, and that Lys8 plays an important role in regulating the gating mode of RyR1.
Seo
In-Ra
IR
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Choi
Mu-Rim
MR
Park
Chul-Seung
CS
Kim
Do Han
DH
D019943:Amino Acid Substitution; D000818:Animals; D002120:Calcium Channel Agonists / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D018482:Muscle, Skeletal / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000502:physiology*; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D012605:Scorpions / Q000737:chemistry / Q000235:genetics
D002120:Calcium Channel Agonists; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012604:Scorpion Venoms; C105361:imperatoxin A