Voltage-Sensing Domain of the Third Repeat of Human Skeletal Muscle NaV1.4 Channel As a New Target for Spider Gating Modifier Toxins.

Voltage-gated sodium channels (NaV) have a modular architecture and contain five membrane domains. The central pore domain is responsible for ion conduction and contains a selectivity filter, while the four peripheral voltage-sensing domains (VSD-I/IV) are responsible for activation and rapid inactivation of the channel. "Gating modifier" toxins from arthropod venoms interact with VSDs, influencing the activation and/or inactivation of the channel, and may serve as prototypes of new drugs for the treatment of various channelopathies and pain syndromes. The toxin-binding sites located on VSD-I, II and IV of mammalian NaV channels have been previously described. In this work, using the example of the Hm-3 toxin from the crab spider Heriaeus melloteei, we showed the presence of a toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. A developed cell-free protein synthesis system provided milligram quantities of isolated (separated from the channel) VSD-III and its 15N-labeled analogue. The interactions between VSD-III and Hm-3 were studied by NMR spectroscopy in the membrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relatively high affinity to VSD-III (dissociation constant of the complex Kd ~6 µM), comparable to the affinity to VSD­I and exceeding the affinity to VSD-II. Within the complex, the positively charged Lys25 and Lys28 residues of the toxin probably interact with the S1-S2 extracellular loop of VSD-III. The Hm-3 molecule also contacts the lipid bilayer surrounding the channel.

CombLabel: rational design of optimized sequence-specific combinatorial labeling schemes. Application to backbone assignment of membrane proteins with low stability.

Assignment of backbone resonances is a necessary initial step in every protein NMR investigation. Standard assignment procedure is based on the set of 3D triple-resonance (1H-13C-15N) spectra and requires at least several days of experimental measurements. This limits its application to the proteins with low stability. To speed up the assignment procedure, combinatorial selective labeling (CSL) can be used. In this case, sequence-specific information is extracted from 2D spectra measured for several selectively 13C,15N-labeled samples, produced in accordance with a special CSL scheme. Here we review previous applications of the CSL approach and present novel deterministic 'CombLabel' algorithm, which generates CSL schemes minimizing the number of labeled samples and their price and maximizing assignment information that can be obtained for a given protein sequence. Theoretical calculations revealed that CombLabel software outperformed previously proposed stochastic algorithms. Current implementation of CombLabel robustly calculates CSL schemes containing up to six samples, which is sufficient for moderately sized (up to 200 residues) proteins. As a proof of concept, we calculated CSL scheme for the first voltage-sensing domain of human Nav1.4 channel, a 134 residue four helical transmembrane protein having extremely low stability in micellar solution (half-life ~ 24 h at 45 °C). Application of CSL doubled the extent of backbone resonance assignment, initially obtained by conventional approach. The obtained assignment coverage (~ 50%) is sufficient for ligand screening and mapping of binding interfaces.

Recombinant Production and Structure-Function Study of the Ts1 Toxin from the Brazilian Scorpion Tityus serrulatus.

An effective bacterial system for the production of ß-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of ß-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.

NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel.

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na+ channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of 13C,15N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na+ channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn2+ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. 15N relaxation data revealed characteristic pattern of µs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+ channels. These results validate structural studies of isolated VSDs of Na+ channels and show possible pitfalls in application of this 'divide and conquer' approach.