Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNaV1.7

Improper function of voltage-gated sodium channels (Na_Vs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na_Vs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na_V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na_V through a systematic structure–activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na_V1.7 (hNa_V1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na_V inhibitors.Modulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na_Vs) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na_V1.1–Na_V1.9, Na_x) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na_Vs have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na_V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1–4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na_V isoforms with nanomolar affinity (5–7). Guanidinium toxins inhibit Na⁺ influx through Na_Vs by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8–10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na_V has been solved to date (11–16). Na_V homology models, constructed based on X-ray analyses of prokaryotic Na⁺ and K⁺ voltage-gated channels, do not sufficiently account for experimental structure–activity relationship (SAR) data (6, 17–20), and the molecular details underlying distinct differences in toxin potencies toward individual Na_V subtypes remain undefined (5, 6, 21–23). The lack of structural information motivates a comprehensive, systematic study of toxin–protein interactions.Open in a separate windowFig. 1.(A) Schematic drawing of 1 bound in the Na_V outer pore as suggested by previous electrophysiology and mutagenesis experiments. Each of the four domains (I, orange; II, red; III, gray; and IV, teal) is represented by a separate panel. (B) Schematic representation of double-mutant cycle analysis and mathematical definition of coupling energy (ΔΔE_Ω). X¹ = IC₅₀(WT⋅STX)/IC₅₀(MutNa_V⋅STX), X² = IC₅₀(WT⋅MeSTX)/IC₅₀(MutNa_V⋅MeSTX), Y¹ = IC₅₀(MutNa_V⋅STX)/IC₅₀(MutNa_V⋅MeSTX), and Y² = IC₅₀(WT⋅STX)/IC₅₀(WT⋅MeSTX).Double-mutant cycle analysis has proven an invaluable experimental method for assessing protein–protein, protein–peptide, and protein–small-molecule interactions in the absence of crystallographic data (Fig. 1B and Fig. S1 and refs. 9, 10, and 24–31). Herein, we describe mutant cycle analysis with Na_Vs using STX and synthetically modified forms thereof. Our results are suggestive of a toxin–Na_V binding pose distinct from previously published views. Our studies have resulted in the identification of a natural variant of STX that is potent against the STX-resistant human Na_V1.7 isoform (hNa_V1.7). Structural insights gained from these studies provide a foundation for engineering guanidinium toxins with Na_V isoform selectivity.Open in a separate windowFig. S1.Mutant cycle analysis definition and examples. (A) Schematic of a single mutant cycle with mathematical expressions for coupling energy ΔΔE_Ω. R is the ideal gas constant and T is temperature. Each IC₅₀ is the half maximal inhibition concentration determined by whole-cell voltage-clamp electrophysiology. When the separation between IC₅₀ values for the reference compound and the modified compound is different with a mutant than with the WT protein, a nonzero value for ΔΔE_Ω is obtained (B), but when the separation is the same (C), ΔΔE_Ω is equal to 0. In B, the difference in the relative affinity of 1 and 4 with Y401A is smaller than the difference with the WT channel, indicating a positive coupling (ΔΔE_Ω > 0). In C, the relative affinities of 1 and 8 against WT rNa_V1.4 and Y401A are similar, and ΔΔE_Ω ∼0 kcal/mol.