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Novel Interactions Identified between µ-Conotoxin and the Na⁺ Channel Domain I P-loop: Implications for Toxin-Pore Binding Geometry

Tian Xue, Irene L. Ennis, Kazuki Sato ^*, Robert J. French  and Ronald A. Li

Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA; ^* Fukuoka Women's University, Fukuoka, Japan; and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada

Correspondence: Address reprint requests to Ronald Li, Assistant Professor of Medicine, Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave./Ross 844, Baltimore MD 21205. E-mail: ronaldli{at}jhmi.edu.

µ-Conotoxins (µ-CTX) are peptides that inhibit Na⁺ flux by blocking the Na⁺ channel pore. Toxin residue arginine 13 is critical for both high affinity binding and for complete block of the single channel current, prompting the simple conventional view that residue 13 (R13) leads toxin docking by entering the channel along the pore axis. To date, the strongest interactions identified are between µ-CTX and domain II (DII) or DIII pore residues of the rat skeletal muscle (Na_v1.4) Na⁺ channels, but little data is available for the role of the DI P-loop in µ-CTX binding due to the lack of critical determinants identified in this domain. Despite being an essential determinant of isoform-specific tetrodotoxin sensitivity, the DI-Y401C variant had little effect on µ-CTX block. Here we report that the charge-changing substitution Y401K dramatically reduced the µ-CTX affinity (300-fold). Using mutant cycle analysis, we demonstrate that K401 couples strongly to R13 (G > 3.0 kcal/mol) but not R1, K11, or R14 (<<1 kcal/mol). Unlike K401, however, a significant coupling was detected between toxin residue 14 and DI-E403K (G = 1.4 kcal/mol for the E403K-Q14D pair). This appears to underlie the ability of DI-E403K channels to discriminate between the GIIIA and GIIIB isoforms of µ-CTX (p < 0.05), whereas Y401K, DII-E758Q, and DIII-D1241K do not. We also identify five additional, novel toxin-channel interactions (>0.75 kcal/mol) in DII (E758-K16, D762-R13, D762-K16, E765-R13, E765-K16). Considered together, these new interactions suggest that the R13 side chain and the bulk of the bound toxin µ-CTX molecule may be significantly tilted with respect to pore axis.