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How Batrachotoxin Modifies the Sodium Channel Permeation Pathway: Computer Modeling and Site-Directed Mutagenesis

Department of Biology, State University of New York at Albany, Albany, New York (S.Y.W.); Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada (D.B.T., B.S.Z.); and Department of Anesthesia, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts (J.M., G.K.W.)

A structural model of the rNav1.4 Na⁺ channel with batrachotoxin (BTX) bound within the inner cavity suggested that the BTX pyrrole moiety is located between a lysine residue at the DEKA selectivity filter (Lys1237) and an adjacent phenylalanine residue (Phe1236). We tested this pyrrole-binding model by site-directed mutagenesis of Phe1236 at D3/P-loop with 11 amino acids. Mutants F1236D and F1236E expressed poorly, whereas nine other mutants either expressed robust Na⁺ currents, like the wild-type (F1236Y/Q/K), or somewhat reduced current (F1236G/A/C/N/W/R). Gating properties were altered modestly in most mutant channels, with F1236G displaying the greatest shift in activation and steady-state fast inactivation (-10.1 and -7.5 mV, respectively). Mutants F1236K and F1236R were severely resistant to BTX after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz), whereas seven other mutants were sensitive but with reduced magnitudes compared with the wild type. It is noteworthy that rNav1.4-F1236K mutant Na⁺ channels remained highly sensitive to block by the local anesthetic bupivacaine, unlike several other BTX-resistant mutant channels. Our data thus support a model in which BTX, when bound within the inner cavity, interacts with the D3/P-loop directly. Such a direct interaction provides clues on how BTX alters the Na⁺ channel selectivity and conductance.

Address correspondence to: Ging Kuo Wang, Department of Anesthesia, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115. E-mail: wang{at}zeus.bwh.harvard.edu

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