Molecular Studies of Alpha-Scorpion Toxin Interactions with Voltage-gated Sodium Channels

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Molecular Studies of Alpha-Scorpion Toxin Interactions with Voltage-gated Sodium Channels

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Title: Molecular Studies of Alpha-Scorpion Toxin Interactions with Voltage-gated Sodium Channels
Author: Wang, Jinti
Abstract: Voltage-gated sodium channels are responsible for initiation and propagation of the action potential in vertebrate nerve and muscle. They are also the molecular targets for a large number of paralytic neurotoxins. Alpha-scorpion toxins, including LqhII (Leiurus quinquestriatus hebraeus, type II), bind to the extracellular domain on the sodium channel and inhibit channel fast inactivation. Their binding prolongs sodium channel opening, leading to repetitive firing, depolarization and conduction block. As a consequence, these toxins can kill organisms by inducing paralysis and cardiac arrhythmia. Using site-directed mutagenesis, we have identified residues that constitute the functional interaction surfaces of alpha-scorpion toxin and its receptor site on the voltage-gated sodium channel. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for LqhII, and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of sodium channels and reveals its mode of interaction with a gating modifier toxin. The bioactive surface of LqhII has recently been shown to be made of a conserved core domain (Phe-15, Arg-18, Trp-38, and Asn-44) and a variable NC domain (Lys-2, Thr-57, Lys-58). In this work, possible interactions on surfaces of alpha-scorpion toxin and its receptor site on the voltage-gated sodium channel were tested by thermodynamic mutant cycle analysis. Single mutations at key amino acid residues important for activity on toxin and sodium channel were constructed by mutagenesis. We have identified an intermolecular interaction between extracellular loop of sodium channel and alpha-scorpion toxins. We demonstrated a specific aromatic-aromatic interaction between amino acid residue Phe1610 and Trp38 of LqhII, a residue that is conserved among many alpha-scorpion toxins. Toxin dissociation was accelerated by depolarization and increased by mutations at both sites, whereas association rates at negative membrane potentials were not changed for mutation at Phe1610, but slightly increased for mutation at Trp38. These results constrain the possible orientation of alpha-scorpion toxin with respect to the gating-module of DIV in sodium channel and suggest that upon interaction, the core-domain of LqhII is in close proximity to the sodium channel. We found that an antianginal and anti-ischemic drug, ranolazine, attenuated sustained Na+ current induced by alpha-scorpion toxin, with a 50% inhibitory concentration (IC50) of 102 ± 10.7 uM. It also attenuated the peak Na+ currents, with an IC50 of 334 ± 2.6 uM. The results demonstrate that ranolazine has antagonist effect against alpha-scorpion toxin. Consistent with this effect on sodium channels, ranolazine reduces the lethal paralytic effects of LqhII in mice.
Description: Thesis (Ph.D.)--University of Washington, 2012
URI: http://hdl.handle.net/1773/20848
Author requested restriction: Delay release for 2 years -- then make Open Access
Date available: 2014-09-03

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