Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

• Published in: The Journal of biological chemistry 2014-08, Vol.289 (31), p.21770-21781
• Main Authors: Lukacs, Peter, Gawali, Vaibhavkumar S., Cervenka, Rene, Ke, Song, Koenig, Xaver, Rubi, Lena, Zarrabi, Touran, Hilber, Karlheinz, Stary-Weinzinger, Anna, Todt, Hannes
• Format: Article
• Language: English
• Subjects: Anesthetic; Anesthetics, Local - pharmacology; Animals; Docking; Ion Channel Gating; Membrane Biology; Molecular Dynamics Simulation; Molecular Modeling; Molecular Pharmacology; Mutagenesis; Mutagenesis, Site-Directed; Patch Clamp Electrophysiology; Patch-Clamp Techniques; Permeability; Protein Conformation; Protein Design; Protein Structure; Sodium Channel; Sodium Channels - chemistry; Sodium Channels - drug effects; Sodium Channels - genetics; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.541763

Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels. Background: Currently the structure of eukaryotic voltage-gated Na+ channels (VGSCs) is predicted from available prokaryotic VGSC crystals. Results: In a mammalian VGSC, the predicted position of a multifunctional tryptophan in the external vestibule enabled design of a second conduction pathway. Conclusion: External parts of pro- and eukaryotic VGSCs are structurally similar. Significance: Engineered external drug access pathways may allow development of novel VGSC modulators.