Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Effects of Recombinant Imperatoxin A (IpTxa) Mutants on the Rabbit Ryanodine Receptor

  • Seo, In-Ra (Department of Life Science, Gwangju Institute of Science and Technology) ;
  • Choi, Mu-Rim (Department of Life Science, Gwangju Institute of Science and Technology) ;
  • Park, Chul-Seung (Department of Life Science, Gwangju Institute of Science and Technology) ;
  • Kim, Do Han (Department of Life Science, Gwangju Institute of Science and Technology)
  • Received : 2006.09.01
  • Accepted : 2006.10.23
  • Published : 2006.12.31
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Abstract

Imperatoxin A ($IpTx_a$), 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, $IpTx_a$ 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 $IpTx_a$ was identical to the synthetic $IpTx_a$ with respect to high-performance liquid chromatography mobility, amino acid composition and specific effects on RyR1. Mutations of certain basic amino acids ($Lys^{19}$, $Arg^{23}$, and $Arg^{33}$) dramatically reduced the capacity of the peptide to activate RyRs. A subconductance state predominated when $Lys^8$ was substituted with alanine. These results suggest that some basic amino acid residues in $IpTx_a$ are important for activation of RyR1, and that $Lys^8$ plays an important role in regulating the gating mode of RyR1.

Keywords

Acknowledgement

Supported by : Korean Ministry of Science and Technology

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