Related Experiment Videos
Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle
N S Chang1, R J French, G M Lipkind
1Department of Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois 60637, USA.
Abstract:
High-affinity mu-conotoxin block of skeletal muscle Na+ channels depends on an arginine at position 13 (Arg-13). To understand both the mechanism of toxin interaction and the general structure of its binding site in the channel mouth, we examined by thermodynamic mutant cycle analysis the interaction between the critical Arg-13 and amino acid residues known to be in the channel's outer vestibule. Arg-13 interacts specifically with domain II Glu-758 with energy of about -3.0 kcal/mol, including both electrostatic and nonelectrostatic components, and with Glu-403 with energy of about -2.0 kcal/mol. Interactions with the other charged residues in the outer vestibule were shown to be almost entirely electrostatic, because these interactions were maintained when Arg-13 was replaced by lysine. These results place the bound Arg-13 at the channel mouth adjacent to the P (pore) loops of domains I and II. Distance estimates based on interaction energies suggest that the charged vestibule residues are in relative positions similar to those of the Lipkind-Fozzard vestibule model [Lipkind, G. M., and Fozzard, H. A. (1994) Biophys. J. 66, 1-13]. Kinetic analysis suggests that Arg-13 interactions are partially formed in the ligand-channel transition state.
Insights
High-affinity mu-conotoxin binding to skeletal muscle sodium channels relies on Arg-13. This residue specifically interacts with channel pore loops, revealing the toxin
Area of Science:
- Biophysics
- Molecular Pharmacology
- Ion Channel Biology
Background:
- High-affinity mu-conotoxin blocks skeletal muscle sodium channels.
- This block is critically dependent on arginine at position 13 (Arg-13).
Purpose of the Study:
- To elucidate the interaction mechanism between mu-conotoxin's Arg-13 and the sodium channel.
- To determine the structural organization of the toxin's binding site within the channel's outer vestibule.
Main Methods:
- Thermodynamic mutant cycle analysis was employed.
- Interactions between Arg-13 and outer vestibule residues were quantified.
Main Results:
- Arg-13 forms specific interactions with domain II Glu-758 (-3.0 kcal/mol) and Glu-403 (-2.0 kcal/mol).
- Interactions with other charged residues are primarily electrostatic.
- Arg-13 is positioned adjacent to pore loops of domains I and II.
- Kinetic analysis indicates partial formation of Arg-13 interactions during the transition state.
Conclusions:
- The study precisely maps the binding site of mu-conotoxin within the skeletal muscle sodium channel.
- Findings support and refine existing models of the channel's outer vestibule structure.
- The results provide insights into the molecular basis of toxin-channel interactions.