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Research Papers

Docking of μ-Conotoxin GIIIA in the Sodium Channel Outer Vestibule

Gaurav Choudhary, Marcela P. Aliste, D. Peter Tieleman, Robert J. French and Samuel C. Dudley, Jr

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μ-Conotoxin GIIIA (μ-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 pore-forming (P) loops, each with an acidic residue near the mouth of the vestibule. μ-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 μ-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 toxin-channel interactions, hitherto undescribed, that may contribute to high-affinity binding and channel isoform selectivity.

Authors

Gaurav Choudhary

Marcela P. Aliste

University of Calgary

D. Peter Tieleman

University of Calgary

Robert J. French

University of Calgary

Samuel C. Dudley, Jr

University of Illinois at Chicago

Purchase article for $19

Subscribe to this journal for $79/year