• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.34-34
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• 1996

Previously we showed that $Ba^{2+}$ block of large conductance $Ca^{2+}$-activated $K^{+}$ (BK) channel was larger in the planar lipid bilayer formed with negatively-charged phosphatidylserine (PS) than neutral phosphatidylethanolamine (PE). In this work, have studied the blocking effect of two $K^{+}$ channel blockers with different mechanisms of action, $Ba^{2+}$ and tetraethylammonium (TEA), on BK channels of rat skeletal muscle. (omitted)itted)

• Molecules and Cells
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• v.22 no.3
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• pp.328-335
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• 2006

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.