Structure-Function Map of the Receptor Site for β-Scorpion Toxins in Domain II of Voltage-gated Sodium Channels

Joel Z Zhang,Vladimir Yarov-Yarovoy,Todd Scheuer,Izhar Karbat,Lior Cohen,Dalia Gordon,Michael Gurevitz,William A Catterall

doi:10.1074/jbc.m111.282509

Joel Z Zhang, Vladimir Yarov-Yarovoy + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111.282509

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Abstract

Voltage-gated sodium (Na(v)) channels are the molecular targets of β-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of β-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.

Highlights

Voltage-gated sodium (Nav)3 channels are responsible for generation and propagation of action potentials in nerves and in skeletal and cardiac muscle cells [1]
We have refined the molecular map of the amino acid residues in these two extracellular linkers that are required for CssIV binding and voltage sensor trapping, and we have discovered a hot spot for toxin action where single mutations in adjacent amino acid residues can either enhance or impair toxin
The Receptor Site for ␤-Scorpion Toxins Includes a Hot Spot in the IIS3-S4 Loop—In the experiments described here, we have substantially extended our previous studies of the molecular determinants of voltage sensor trapping (8 –10) and further established that both IIS1-S2 and IIS3-S4 loops are required for normal binding and action of ␤-scorpion toxins, whereas the IIS3-S4 loop plays a dominant role

Summary

EXPERIMENTAL PROCEDURES

PCR-directed Mutagenesis—Mutations were introduced by site-directed mutagenesis using a PCR-based strategy as described previously [9]. All data are presented as mean Ϯ S.E. Structural Modeling—Homology and de novo modeling of the voltage sensor in domain II of rat Nav1.2a channels was performed using the Rosetta-Membrane method [3, 12]. Docking simulations of CssIV binding to the voltage sensor in domain II of the rNav1.2a channel were performed using the Rosetta docking method [16, 17]. 10,000 models were generated and the best model was chosen among 20 lowest scoring models as the model that fit the majority of available experimental data on key residues contributing to interaction of the CssIV toxin with voltagesensing domain II of rat Nav1.2a channels [8, 9, 18] Backbone flexibility of the extracellular part of the voltage sensor (residues Cys768-Gly800 and Ser832-Arg856) was allowed during simulations and Tyr and Tyr residues of the ␤-scorpion CssIV toxin were required to be at the voltage sensor-toxin interface. 10,000 models were generated and the best model was chosen among 20 lowest scoring models as the model that fit the majority of available experimental data on key residues contributing to interaction of the CssIV toxin with voltagesensing domain II of rat Nav1.2a channels [8, 9, 18]

RESULTS

Voltage dependence of activation

Slope n

DISCUSSION