Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of Na+ Channel Voltage Sensors in Domains II and IV

Abstract

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxinbinding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.Voltage-gated sodium channels (VGSCs) 3 play critical roles in the generation and propagation of action potentials. Nine VGSC ␣ subunit subtypes (Nav1.1-1.9) have been cloned and characterized from mammals (1). These subtypes are expressed in different excitable tissues and are involved in distinct physiological functions such as neurotransmitter release, muscle contraction, secretion, and pain sensation (2-4). The nine ␣ subunits share a common four-domain (DI-DIV) structure, in which each domain has six transmembrane segments (S1-S6) (1). Based on distinct functional behaviors during channel activity, the six transmembrane segments are generally separated into two structural components (5). The central pore module is the basis for the ion conduction pathway and is formed by the S5-S6 region of the channel. The voltage sensor modules are formed by the S1-S4 regions and are essentially independent structures that directly respond to changes in the transmembrane potential with conformational alterations that are coupled to opening and closing of the pore module. VGSCs undergo voltage-dependent activation subsequently followed by fast inactivation through successive activities of the voltage sensors in the four domains. The S4 segments in the voltage sensors of DI, DII, and DIII are determinants of channel activation, whereas that of DIV is predominantly involved in channel inactivation (5-8). The four membrane-spanning segments (S1-S4) of the voltage sensors of mammal VGSCs exhibit high sequence similarity in the four domains, but the amino acids and sequenc...

Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)

“…More subtle and primarily subtype-specific reductions in activity were observed with K18A, R26A, K27A, and R29A. Arg 26 , Lys 27 , and Arg 29 are located on loop 4 of HwTX-IV, and our model predicts that some of these amino acids may interact with acidic residues, such as Asp 816 and Glu 818 , located on the DII S3-S4 linker of Nav1.7, that have previously been identified as important for HwTX-IV activity (19). Two of these basic residues (Lys 27 and Arg 29 ) have also been shown to be important for block of VGSCs in rat DRG neurons by the closely related spider toxin, hainantoxin IV (-TheraphotoxinHhn1b) (20).…”

“…6b). In addition, our docking model suggests a likely interaction of basic Lys 32 with acidic Glu 811 in Nav1.7 (Glu 837 in Nav1.2), an attractive model given the highly disruptive nature of the K32A and E811Q mutations individually compared with other peptide and channel mutations (19). Interestingly, according to our model, P11A, D14A, L22A, and K18A are located on loops 2 and 3, facing away from the groove formed by the Nav1.7 S1-S2 and S3-S4 loops.…”

“…S4 was manually moved down into a resting state configuration, the S1-S2 and S3-S4 loops were regenerated, and the entire model was energy-minimized. Native HwTX-IV was manually docked into the Nav1.7 homology model based on the results of the alanine scan of HwTX-IV inhibition against Nav1.7 and on published Nav1.7 mutations that affect HwTX-IV binding (8,18,19). Following the manual docking, the entire Nav1.7 DII S1-S4 with docked HwTX-IV system was minimized, and an implicit membrane molecular dynamics simulation was performed using the CHARMm force field with Generalized Born Implicit Membrane (Discovery Studio (36)) to further refine the docked structure.…”

“…However, very little is known about the channel residues involved in these interactions. The only studies addressing this question are from Cummins and colleagues (8,18,19), who have identified several residues in the S3-S4 linker region of domain II that are crucial for huwentoxin-IV inhibition of Nav1.7. Even less is known about the toxin residues that interact with VGSCs.…”

Gating-Pore Currents Demonstrate Selective and Specific Modulation of Individual Sodium Channel Voltage-Sensors by Biological Toxins

“…Selective Voltage-Sensor Modulation by Biological Toxins used extensive site-directed mutagenesis to show that tarantula toxin HWTX-IV inhibition of central-pore currents critically depends on specific residues in the DII voltagesensor (Xiao et al, 2008;Xiao et al, 2011), suggesting that HWTX-IV indirectly inhibited central-pore currents by blocking movement of a voltage-sensor, and thus preventing activation of the channel. We examined the effects of HWTX-IV on inward gating-pore currents generated by each of the four voltage-sensors and found that HWTX-IV only significantly altered the gating-pore currents generated in DII (Fig.…”