m-Conotoxin GIIIA (m-CTX) is a high-affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na+ channel. The detailed bases for the toxin's high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four poreforming (P) loops, each with an acidic residue near the mouth of the vestibule. m-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na+ channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of m-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.
Bibliographical noteFunding Information:
Gaurav Choudhary is supported by a Career Development Award from the BLRGD Service, Department of Veteran Affairs. D. Peter Tieleman is an AHFMR Senior Scholar and CIHR New Investigator. This study was supported by a National Institutes of Health (NIH) grants, HL64828 and HL73753 (Samuel C. Dudley, Jr.), a Department of Veterans Affairs Merit grant (Samuel C. Dudley, Jr.), and an American Heart Association Established Investigator Award (Samuel C. Dudley, Jr.). Robert J. French is an AHFMR Medical Scientist, and was supported by operating grants from CIHR, and from HSF of Alberta, NWT & Nunavut. We thank Dr. Denis McMaster, Peptide Services, Faculty of Medicine, University of Calgary, for producing the conotoxin derivatives used, and Drs. Harry Fozzard and Gregory Lipkind, University of Chicago, for providing the initial outer vestibule model and for helpful discussions.
- Computer simulation
- Ion channel
- Molecular model
- Mutational analysis
- Structural biology