. Author manuscript; available in PMC: 2026 Sep 19.

Published in final edited form as: Cell Chem Biol. 2026 Sep 1;33(9):1322–1332.e7. doi: 10.1016/j.chembiol.2026.05.016

A designer saxitoxin-engineered sodium channel pair as a general tool for subtype-selective inhibition

Elizabeth R Park ¹, Nicholas Denomme ², Holly S Hajare ¹, J Du Bois ^1,^3,^*

PMCID: PMC13537208 NIHMSID: NIHMS2193175 PMID: 42679814

SUMMARY

We have used small molecule chemical design and protein engineering to fashion an orthogonal ligand-channel pair that enables acute and selective inhibition of a genetically encoded voltage-gated sodium channel (Na_V). A modified ligand based on the nerve toxin, saxitoxin (STX), binds potently and reversibly to a Na_V bearing two unique amino acid mutations in the outer mouth of the channel (Site 1). The same ligand is ineffective at blocking wild-type Na_Vs and does not disrupt action potential signals in neuronal cells or brain tissue at working concentrations. The versatility of this tool derives from the highly conserved outer pore (Site 1), as the same two amino acid replacements can be introduced in Na_V1.1–1.4, 1.6, or 1.7, thus sensitizing each channel to inhibition by the designer STX ligand. This tool will empower studies of Na_V-isoform physiology to better understand the role of Na_Vs in health and disease.

Keywords: Chemogenetics, designer ligand, protein mutagenesis, electrophysiology, voltage-gated sodium channel

ETOC BLURB

Park, et al. describe a high precision, small molecule tool patterned after a natural toxin for acute, selective, and reversible inhibition of an engineered voltage-gated sodium channel. By combining chemistry and genetics, this technology enables physiological studies of any one of six Na_V subtypes (Na_V1.1–1.4, 1.6, 1.7).

Graphical Abstract

graphic file with name nihms-2193175-f0008.webp

INTRODUCTION

The nervous system is a highly complex, interconnected network of specialized cells that rapidly process and transmit information through chemical and electrical signaling. Electrical impulses (action potentials, APs) arise as frequency- and amplitude-modulated spike trains resulting from the choreographed interactions of ion channels.¹ Among the collection of channels responsible for AP signaling, voltage-gated sodium channels (Na_Vs) provide the explosive inward sodium current that drives AP firing and propagation.^1–3 In mammalian cells, sodium influx is carried by nine isoforms of the pore-forming α-subunit, Na_V1.1–1.9, each a ~260 kDa protein with four nonidentical repeats, Domains I–IV (DI–DIV), and co-expressed with one or two auxiliary β-subunits.⁴ Na_V isoforms are further divided into tetrodotoxin (TTX)-sensitive (Na_V1.1–1.4, Na_V1.6, Na_V1.7) and TTX-resistant (Na_V1.5, Na_V1.8, Na_V1.9) channels based on whether IC₅₀ values for TTX fall below or exceed 1 μM.⁵ Transcriptional splicing, editing, and post-translational modifications further diversify the population of Na_V proteoforms expressed in the plasma membranes of electrically excitable cells.^6–11

Na_V malfunction is causally linked to a number of human pathologies including arrhythmia, chronic pain, epilepsy, and neurodevelopmental disorders.^12–14 A desire to understand how specific Na_V subtypes shape APs and how Na_V dysregulation underlies disease etiology motivates the work described herein. Recent advances notwithstanding, developing pharmacological tools that target individual Na_V subtypes is challenged by the highly conserved nature of the nine α-subunits and the added structural variability introduced through post-transcriptional and post-translational changes.^6,7,15 In addition, cell type-selective differentiation of a single Na_V isoform is not possible with molecules that target wild-type (WT) channels. An approach that combines chemistry and genetics, analogous to DREADDs and RASSLs for GPCRs or PSAMs for nAChRs,^16–18 offers an enticing solution—one that promises rapid and reversible control of a specifically encoded Na_V. We report a universal chemogenetic tool for selective inhibition of TTX-sensitive Na_Vs. TTX-sensitive channels appear in the CNS, in cardiac and skeletal muscle, and in non-electrically excitable cells (e.g., lymphocytes, glia).^19,20 Accordingly, this technology will enable precise pharmacological interrogation of Na_V-subtype physiology and, given the obligatory role of Na_Vs in AP signaling, offers a complementary method to optogenetics for focally-defined block of neuronal activity.

DESIGN

The highly conserved locus of amino acids that define the outer pore and selectivity filter in Na_Vs offers an ideal receptor site (Site 1) for chemogenetic ligand design (Fig. 1, S1). Organisms that concentrate Site 1 guanidinium toxins, as exemplified by TTX and saxitoxin (STX), introduce select mutations to the outer pore as a resistance mechanism.^21–24 These structural changes do not result in an evident fitness cost nor do they alter gating. As for ligands that target the pore, STX, which binds rapidly, reversibly, and with low nanomolar affinity to TTX-sensitive channels and is accessible along with derivative forms through de novo synthesis, is an optimal lead.^25,26 The overwhelming majority of small molecules that modulate Na_Vs are state-dependent and display slow association and/or dissociation kinetics.^27–34 For these reasons, our studies have focused on compensatory modifications to Site 1 and STX to generate a chemogenetic ligand-channel pair.

Figure 1. — (A) Structure of hNa_V1.7 with STX bound in the outer vestibule (Site 1). Reproduced from PDB 6J8G.⁶¹ DI = orange, DII = red, DIII = gray, DIV = cyan. The ion selectivity filter is denoted in yellow.

(B) Sequence alignment of outer pore residues across rNa_V1.1–1.9. Ion selectivity filter residues are bolded. Residues that destabilize STX binding to Na_V1.5, 1.8, and 1.9 are in blue. See also Fig. S1.

RESULTS

STX-amide derivatives are broadly ineffective at inhibiting wild-type Na_Vs

For our initial prototype, we targeted a designer molecule that would inhibit an engineered Na_V with nanomolar potency (IC₅₀ < 100 nM) and >100:1 selectivity relative to WT channels. To identify a starting ligand, we compared STX and STX C13-derivatives that included a naturally occurring acetate ester (LWTX-5), an analogous acetamide 1, and a small collection of aryl amides.^25,35 Half-maximal inhibitory concentrations (IC₅₀) for STX and LWTX-5 against WT rat Na_V1.4 (rNa_V1.4) α-subunit expressed in Chinese hamster ovary K1 (CHO-K1) cells are 2.9 ± 0.1 and 83 ± 3.9 nM, respectively.²⁵ A marked decrease in potency, however, is noted across the amide series (Fig. 2, S2), including acetamide 1 (IC₅₀ = 1.9 μM). As with STX, channel block by these amide derivatives is fast and reversible. The >500-fold change in IC₅₀ values for these compounds is not explained by available structural and SAR data and may stem from relative differences in solvation energies between the unbound and bound ligand and the energetic cost of desolvating the amide, carbamate, or ester moiety.^36–38 The reason(s) for such potency differences aside, these modified toxins, particularly aryl amides such as STX-NBz 2, offer an attractive starting point for chemogenetic ligand design. The lack of potency for STX-NBz 2 manifests beyond rNa_V1.4 and includes all TTX-sensitive (Na_V1.1–1.4, 1.6, 1.7) and TTX-resistant (e.g., Na_V1.5) channels (Fig. 2C).

Figure 2. — (A) Structures of STX, LWTX-5, and non-natural STX-amides.

(B) Electrophysiological characterization of STX-amides against WT rNa_V1.4. IC₅₀ ± s.e.m.

(C) 1 μM STX-NBz 2 was exposed to Na_V1.1–1.7 to determine cross-isoform potency. Data represent percent reduction in initial current in comparison to baseline, mean ± s.e.m., n ≥ 3. The IC₅₀ for 2 is estimated to be ≥1 μM for each subtype tested as noted by the dashed red line.

STX-amide potency is rescued through rNa_V1.4 outer pore mutations

To explore mutations that rescue the potency of STX-amides, single amino acid substitutions were made across DI–DIV in the p-loop α-helices comprising the STX binding site. Because the engineered Na_V must retain WT channel function, we opted for conservative mutations in the outer mouth of Na_Vs, largely targeting residues that vary in species resistant to guanidinium toxins. Mutations were introduced into Scn4a, the gene encoding rNa_V1.4, and the corresponding channels were successfully expressed in CHO-K1 cells. Mutant Na_Vs were tested only if they displayed current densities and gating properties that closely mirrored WT rNa_V1.4. In the first-pass analysis, the potency of STX-NBz 2 was evaluated against mutant channels at 1 μM (Fig. S3). Known mutations that destabilize STX binding—Y401C, Y401S, E403D, E758D, and D1532N—also disrupted block by STX-NBz 2, as no recordable inhibition of these channels by 1 μM 2 was measured.^25,39–41 On the other hand, substitution of D1241 with hydrophobic residues (e.g., Leu, Ile) improved STX-NBz 2 potency (Fig. S3) but decreased the affinity of STX (Fig. S4). Unexpectedly, block of rNa_V1.4 D762E and G1533A mutants by STX-NBz 2 and STX was enhanced relative to WT channel (Figs. S3, S4); these mutations may similarly influence the binding of other bis-guanidinium toxins.⁴² Concentration-response curves with STX-NBz 2 and four rNa_V1.4 mutant channels, D762E, D1241L, D1241I, and G1533A, show that the potency of STX-NBz 2 is improved 10-fold by the most stabilizing mutation, Domain III D1241L (WT rNa_V1.4: IC₅₀ = 1610 ± 45 nM; D1241L: 163 ± 8 nM, Fig. 3).

Figure 3. — (A) PDB 6J8G (hNa_V1.7 with STX bound) highlighting the location of STX-NBz 2 stabilizing mutations.⁶¹ DI = orange, DII = red, DIII = gray, DIV = cyan. Residues D762 (DII), I1241 (DIII), and G1533 (DIV) are represented in yellow space filling. Note that residue I1241 appears as D1241 in rat Na_V1.7 (see Fig. S1). For additional views of the channel pore, see Data S1.

(B) Electrophysiological characterization of STX-NBz 2 against transiently expressed rNa_V1.4 wild-type and select mutant channels. IC₅₀s ± s.e.m. (nM), Hill coefficients ± s.e.m.: rNa_V1.4 = 1610 ± 45, −1.03 ± 0.03; D762E = 357 ± 31, −1.04 ± 0.10; G1533A = 235 ± 11, −1.21 ± 0.07; D1241I = 279 ± 14, −0.97 ± 0.05; D1241L = 163 ± 8, −0.94 ± 0.05. Graph represents mean ± s.e.m. (rNa_V1.4, n = 3; D762E, n = 4; G1533A, n = 5; D1241I, n = 3; D1241L, n = 3). See also Figs. S3–5.

To further boost the potency of STX-NBz 2 as a channel blocker, we investigated Na_Vs in which two stabilizing mutations from different domains were combined in one construct. Of the double mutant channels evaluated, rNa_V1.4 D762E/G1533A, D762E/D1241L, and D1241L/G1533A were particularly effective in rescuing the potency of the benzamide ligand (Fig. 4A and B). Measured IC₅₀ values for STX-NBz 2 against these three mutant channels range between 35–49 nM. We selected the two top performing constructs, rNa_V1.4 D762E/D1241L and D1241L/G1533A, for subsequent studies. Importantly, introducing either of these pairs of mutations does not alter rNa_V1.4 functional properties compared to WT, including voltage dependence of activation, inactivation, and ion selectivity (Fig. S5, S6). Inhibition of these double mutant channels by STX-NBz 2 is favored over WT rNa_V1.4 by ~45:1.

Figure 4. — (A) Concentration-response curves of STX-NBz 2 against rNa_V1.4 double mutant channels. IC₅₀s ± s.e.m. (nM), Hill coefficients ± s.e.m.: rNa_V1.4 = 1610 ± 45, −1.03 ± 0.03; D762E/G1533A = 49.3 ± 3.6, −0.84 ± 0.06; D762E/D1241L = 37.6 ± 2.2, −1.01 ± 0.06; D1241L/G1533A = 35.3 ± 2.5, −1.06 ± 0.08. Graph represents mean ± s.e.m. (rNa_V1.4, n = 3; D762E/G1533A, n = 3; D762E/D1241L, n = 5; D1241L/G1533A, n = 4).

(B) Representative voltage-clamp traces of WT rNa_V1.4, D762E/D1241L and D1241L/G1533A with 250 nM of STX-NBz 2 applied. Traces recorded in the order: Initial, 250 nM, Wash. Scale bars represent 2 nA or 0.5 nA and 1 ms.

(C) Representative voltage-clamp traces of WT rNa_V1.4 and D1241L/G1533A with 250 nM or 500 nM of STX-N^mTl 4 applied. Traces recorded in the order: Initial, 250 nM, 500 nM, Wash. Scale bars represent 0.5 nA and 1 ms.

(D) Electrophysiological characterization of all STX-amides against rNa_V1.4 double mutant channels, D762E/D1241L and D1241L/G1533A, reported as IC₅₀ ± s.e.m. (nM). Fold change is determined by comparison to WT IC₅₀ values appearing in Figure 2. (1 vs. D762E/D1241L, n = 3; 1 vs. D1241L/G1533A, n = 4; 3 vs. D762E/D1241L, n = 3; 3 vs. D1241L/G1533A, n = 4; 4 vs. D762E/D1241L, n = 4; 4 vs. D1241L/G1533A, n = 10; 5 vs. D762E/D1241L, n = 4; 5 vs. D1241L/G1533A, n = 3; 6 vs. D762E/D1241L, n = 3; 6 vs. D1241L/G1533A, n = 3). nd = not determined. See also Figs. S2, S5 and S6.

Ligand modification further improves selectivity for mutant channels

We measured IC₅₀ values for additional STX-amide derivatives (1, 3–6) against rNa_V1.4 D762E/D1241L and D1241L/G1533A mutants to determine if ligand substitution could bolster the selectivity factor for block versus the WT channel. Among these five ligands, meta-toluamide, STX-N^mTl 4, displayed >100:1 selectivity for rNa_V1.4 D1241L/G1533A over WT and a favorable IC₅₀ of 39.1 ± 1.7 nM (Fig. 4C and D). Other C13-amide ligands such as ortho-toluamide, STX-N°Tl 5, and cyclohexylcarboxamide, STX-NCy 6, were ineffective channel blockers (IC₅₀ > 1 μM) or fell short of our targeted 100:1 selectivity (STX-NAc 1, para-toluamide STX-N^pTl 3) (Fig. S2). The combination of rNa_V1.4 D1241L/G1533A and STX-N^mTl 4 satisfies our original design criteria for a potent and selective chemogenetic tool.

STX-N^mTl displays similar selectivity for all TTX-‘sensitive’ Na_Vs

Given the nearly identical sequence structure of the outer pore-forming p-loop repeats across all Na_V subtypes (Fig. 1), we assessed the potency of STX-N^mTl 4 against structurally equivalent D/L (DIII)–G/A (DIV) double mutants of Na_V1.1–1.3 and 1.5–1.7. Sodium current recordings of STX-N^mTl 4 with WT Na_V1.1–1.3 and 1.5–1.7 in CHO-K1 cells confirmed that IC₅₀ values for this compound are >2 μM (Fig. 5). As with rNa_V1.4, introducing D/L–G/A mutations to Na_V1.1–1.3, 1.6, and 1.7 significantly improves the potency of STX-N^mTl 4, reducing IC₅₀ values between 69–149-fold. With the TTX-resistant channel, Na_V1.5, binding of 4 to Na_V1.5 D1423L/G1715A is greatly favored over the WT channel, but the potency of this ligand for the double mutant is low (IC₅₀ = 2.4 μM, Fig. S7). Introducing the D/L–G/A mutants to any one of these subtypes does not alter the biophysical properties of the channel (Fig. S8). Collectively, our electrophysiology data indicate that, in cells expressing both the double mutant and WT Na_Vs, concentrations of STX-N^mTl 4 between 250–500 nM should inhibit a large percentage of mutant channels (73–90%) with minimal block of WT conduction (<10%). Like STX, channel block by STX-N^mTl 4 is kinetically fast (k_on ~10⁶ M⁻¹ s⁻¹), state-independent, and fully reversible (k_off ~10⁻² s⁻¹) (Fig. S9).

Figure 5. — (A) Concentration-response curves of STX-N^mTl 4 with TTX-sensitive WT and double mutant Na_V1.1–1.7 transiently expressed in CHO-K1 cells. The operative concentration range (250–500 nM) is depicted in gray. Data represents mean ± s.e.m. (hNa_V1.1, n = 4; hNa_V1.1 D/L–G/A, n = 3; rNa_V1.2, n = 4; rNa_V1.2 D/L–G/A, n = 6; rNa_V1.3, n = 4; rNa_V1.3 D/L–G/A, n = 5; mNa_V1.6, n = 4; mNa_V1.6 D/L–G/A, n = 4; rNa_V1.7, n = 3; rNa_V1.7 D/L–G/A, n = 5). See also Figs. S7 and S8.

(B) Table of fold change in IC₅₀s comparing WT to double mutant Na_V1.1–1.7. IC₅₀s ± s.e.m.; nd = not determined.

Operating concentrations of STX-N^mTl do not alter AP signaling in cultured neurons or tissue slice

To ensure that operating concentrations (250–500 nM) of STX-N^mTl 4 do not perturb WT Na_V function in primary cells, we applied this ligand to cultured E17–18 rat hippocampal neurons. Sodium current measurements of 4 with hippocampal neurons (days in vitro, DIV 6–8) reveal an IC₅₀ of 3.72 ± 0.26 μM (Fig. 6A and B), a value congruent with CHO-K1 cell data and consistent with the expression of multiple TTX-sensitive isoforms (Na_V1.1–1.3, Na_V1.6) in these cells.^43–45

Figure 6. — (A) Representative voltage-clamp traces of rat E17–18 hippocampal cells (DIV 6–8) treated with increasing concentration of STX-N^mTl 4. Traces collected in the order: Initial, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM. Scale bars represent 1 nA and 1 ms.

(B) Concentration-response curve of STX-N^mTl 4 in embryonic hippocampal neurons. IC₅₀ ± s.e.m. (nM), Hill coefficient ± s.e.m.: 3720 ± 262, −0.95 ± 0.06. Graph represents mean ± s.e.m. (n = 7).

(C) Representative single AP traces of rat E17–18 hippocampal cells (DIV 9–13) with 500 nM or 1 μM of 4 applied. Traces recorded in the order: Initial, 500 nM or 1 μM, Wash. Scale bars represent 20 mV and 5 ms.

(D) Characteristics of a single elicited action potential with 250 nM, 500 nM, or 1 μM of 4 applied to embryonic hippocampal neurons. Amplitude was recorded as peak mV. Rise rate is recorded as the maximum dV/dt and FWHM represents the full width at half maximum amplitude. Data represent mean ± s.e.m. Data are not statistically significantly different as determined by paired t-tests to baseline values. (For 250 nM, n = 8; 500 nM, n = 5; 1 μM, n = 4).

(E) Representative phase plot of a single elicited action potential by an embryonic hippocampal neuron pre-, during, and post-application of 1 μM of STX-N^mTl 4. Traces recorded in the order: Initial, 1 μM, Wash.

Current-clamp electrophysiology experiments with rat hippocampal neurons (DIV 9–13) were performed using four concentrations of STX-N^mTl 4 to determine the influence of this ligand on AP signaling. In these experiments, we evaluated single AP amplitude, half-width, maximum rise rate, and AP train firing rate. Single APs were elicited with a 25 ms injection of +100 pA current. At 1 μM 4, AP amplitude, half-width, and rise rate were unchanged relative to untreated cells (Fig. 6C–E) even though at this concentration, ~30% of WT channels are likely blocked (estimated from voltage-clamp data). This finding accords with previous reports that ~33% of Na_Vs expressed in neurons can be inhibited without altering Na_V-dependent single AP properties.⁴⁶

Action potential trains were elicited through 750 ms injections of +50–100 pA current. Because repetitive firing requires a reserve of available Na_Vs, the AP firing rate is more sensitive to sodium channel block and >10% inhibition can result in a reduction in firing frequency.^46–49 Application of 250 nM STX-N^mTl 4 had no observable influence on AP firing rates (Fig. 7A–C). Increasing 4 to 500 nM concentration results in an 18% diminution in AP firing rate. Only at 1 μM (~30% channels blocked) is AP frequency visibly diminished; full ablation of AP trains is observed at 5 μM 4 (~60% channels blocked). At these concentrations, the effect of 4 on AP firing is completely reversible upon perfusion of cells with external solution.

Figure 7. — (A) Schematic of a whole-cell recording on cultured embryonic hippocampal cells.

(B) Representative current-clamp traces of rat E17–18 hippocampal cells (DIV 9–13) with 250 nM, 500 nM, 1 μM, or 5 μM STX-N^mTl 4 applied. Traces recorded in the order: Baseline, STX-N^mTl 4, Wash. Each concentration of 4 was examined on a different cell. A dotted line at 0 mV is provided for reference. Scale bars represent 20 mV and 100 ms.

(C) Changes in AP firing frequency in an AP train with 250 nM, 500 nM, 1 μM, or 5 μM 4 applied. Data represent mean ± s.e.m. Statistics were calculated with a paired t-test compared to baseline firing frequency, * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. (250 nM, n = 8; 500 nM, n = 10; 1 μM, n = 4; 5 μM, n = 5).

(D) Schematic of whole-cell recording configuration in M1 L5 pyramidal neurons.

(E–G) Effects of 250 nM 4 on normalized firing frequency evoked by 200 pA, 300 pA, or 400 pA. Data represents mean ± s.e.m. (N = 3, n = 4).

(H) Representative AP trains evoked at 200, 300, or 400 pA from the same cell during baseline (black) and 250 nM 4 (blue) conditions. A dotted line at 0 mV is provided for reference. Scale bars represent 20 mV and 1 s.

To further evaluate the working concentration of STX-N^mTl 4, we performed whole-cell current-clamp recordings on layer 5 pyramidal neurons in mouse primary motor cortex (M1). Action potential trains were evoked with somatic current injections (200–400 pA) before, during, and after acute perfusion of 4. At 250 nM, 4 exhibits no effect on input resistance or evoked firing frequency (Fig. 7D–H), in keeping with our findings with cultured neurons. Based on our recordings from both hippocampal cultures and cortical slice preparations, 250 nM 4 should serve as a useful operative concentration for both in vitro and ex vivo electrophysiology experiments, a concentration at which 73–86% of the engineered channels are blocked.

DISCUSSION

The initiation and propagation of APs require the coordinated action of multiple Na_V subtypes. A desire to understand how specific channel isoforms contribute to AP signaling and how AP signaling is altered when subtype trafficking, localization, and/or gating is dysregulated necessitates the invention of high-precision pharmacological tools for altering Na_V function. Additionally, the importance of Na_Vs as therapeutic targets to treat peripheral and central nervous system disorders motivates a broader interest in Na_V physiology and pathophysiology and inspires the studies herein.^20,34,50 In this report, we disclose a chemogenetic mutant Na_V-ligand pair that offers a general solution for selectively manipulating single Na_V subtypes. Future applications of this unique tool are manifold for elucidating Na_V subtype function in different cell types and for pharmacologically modulating APs in engineered neuronal cells and tissue.

The advancement of a ‘one-size-fits-all’ designer ligand-Na_V mutant combination to interrogate the physiology of individual channel isoforms follows from the availability of bis-guanidinium toxin derivatives through de novo synthesis and the highly conserved outer vestibule of Na_V subtypes. Saxitoxin is an optimal template for chemogenetic ligand design given its desirable pharmacodynamic properties (i.e., reversible and state-independent channel binding). The amenability of the STX binding site (Site 1) to select mutations that cause little to no evident perturbation to channel function is equally critical to the advancement of this work.

We have identified three amino acids D762 (DI), D1241 (DIII), and G1533 (DIV) (rNa_V1.4 numbering) that, when appropriately mutated, stabilize binding of STX-N^mTl 4 and related amide structures. When evaluated against WT Na_V1.1–1.4, 1.6, and 1.7 (TTX-sensitive channels), inhibition of double mutant D/L (DIII)–G/A (DIV) constructs by STX-N^mTl 4 is 69–149x more selective. In addition to its potency (IC₅₀ values between 24–81 nM), binding of 4 to Na_Vs is fast and fully reversible. At 250–500 nM concentrations of 4, levels that are near saturation block of the D/L–G/A double mutant (73–90%), the influence of this ligand on WT channels in CHO-K1 cells, primary cultured neurons, and mouse brain slice is effectively nil.

Like STX, STX-N^mTl 4 is a state-independent channel antagonist. This property is advantageous as it enables block of Na_V conductance without stimulation using electrophysiological protocols or pharmacological agents (e.g., peptide venoms) to access different conformational states of the channel. Channel block occurs within seconds upon application of 4, thereby allowing acute and specific inhibition of mutant Na_Vs in cells. A related approach that makes possible differentiation of Na_V1.2 or Na_V1.6 from other Na_V isoforms relies on select mutations in the DIV voltage-sensing domain and a state-dependent arylsulfonamide compound (ASC).^51,52 Mutant channel block by the ASC, however, is incomplete and requires an intricate voltage protocol to prolong depolarization of the cell. These constraints and the use-dependent activity of such ligands can complicate interpretation of the electrophysiological data. The STX-N^mTl–Na_V D/L–G/A double mutant pair is advantaged in this regard.

The ability to block sodium conductance resulting from a single Na_V isoform in a specific cell type should empower studies of cellular, circuit, and network physiology. Modern genetic methods make possible cell type-specific knock-in of D/L–G/A double mutants for any TTX-sensitive WT Na_V channel of interest including those present in glial, cancer, or other non-excitable cells. Differentiating between Na_V isoforms expressed in more than one cell type is not possible with inhibitors such as PF-05089771 or VX-548,^33,53,54 which target WT channels—only a chemogenetic tool makes such precision possible. While experiments involving genetic knock-out or knock-down of Na_Vs have provided valuable insights into subtype function, compensatory effects (i.e., upregulation of an alternative isoform) often obscure results.^55–58 The introduction of point mutations at Site 1 to a TTX-sensitive WT Na_V, however, should have little to no perturbative effect on channel expression and cellular function.

As TTX-sensitive Na_Vs are ubiquitously expressed in CNS tissue and obligatory for AP signaling, fast and reversible pharmacological channel block will enable frequency and amplitude modulation of electrical signals in genetically-defined neurons that comprise a specific neural circuit. Opportunities to interrogate the role of Na_V subtypes in glia and how selective modulation of these channels alter APs are also possible. Ultimately, we expect chemogenetic, STX-based tools to facilitate efforts to understand how Na_V subtypes orchestrate electrical signaling and signal integration in development and disease.

LIMITATIONS OF THIS STUDY

An understanding of the structural details responsible for the ca. 100-fold selectivity of STX-N^mTl 4 for the double mutant over WT Na_Vs remains unresolved at present. We speculate that the two amino acid mutations, D1241L and G1533A, may allow for waters of hydration to intervene in the ligand bound complex in a manner that is not possible in WT Na_Vs. The imaging resolution of cryo-EM is insufficient to substantiate this hypothesis. In the future, ligand binding isotope studies using D₂O solvent could give evidence of water coordination between 4 and the receptor site.^59,60 Molecular dynamic simulations of ligand-channel binding may also help illuminate the origin of selectivity.³⁶

Our study uses Scna genes from different source organisms given their availability and the efficiency with which the corresponding Na_V subtypes express in heterologous cells. Based on the highly conserved sequences of the residues comprising Site 1 between orthologous channels (Fig. S1) and the data we present, translation of our findings to mouse, rat, and/or human channels not employed in this study is expected. Experiments in cells or tissues either transiently or stably expressing the mutant Na_V channel may require calibration owing to differences in IC₅₀ values between different isoforms and orthologues. The variance we observe in IC₅₀ values for STX-N^mTl 4 across different Na_V subtypes from different species offers a useful benchmark in this respect. Ultimately, implementation of our chemogenetic tool to investigate Na_V subtype physiology will require the use of genetic engineering methods to generate knock-in cell lines or transgenic animals. Viral delivery of Scna genes is hindered by the cargo capacity of widely used viral vectors such as AAVs. Efforts to identify a lentiviral delivery system to overcome this limitation are currently ongoing.

RESOURCE AVAILABILITY

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Justin Du Bois (jdubois@stanford.edu).

Materials availability

All plasmids and small molecules generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

Data: All data reported in this paper will be shared by the lead contact upon request.
Code: This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Chinese Hamster Ovary (CHO) Cells

Chinese hamster ovary-K1 (CHO-K1) cells (CCL-61^™) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cell lines were not further authenticated. Cells were grown on 10-cm tissue culture dishes in F12-K medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 50 U/mL penicillin-streptomycin (Thermo Fisher). Cells were maintained at 37 °C with 5% CO₂ and 96% relative humidity and passaged approximately every 3–4 days according to previously described protocols.⁴⁷ Passaging of cells was accomplished as follows: 1) media was aspirated; 2) washed with phosphate buffered saline (PBS); 3) treated with 1 mL of trypsin-EDTA (0.05% trypsin, Millipore Sigma, St. Louis, MO) until completely dissociated (~5 minutes); and 4) diluted with 4 mL of growth medium. Cells were typically passaged at 1:20 to 1:10 dilution.

For electrophysiology, CHO-K1 cells were grown in 6- or 24-well tissue culture dishes and generally transfected at 40–80% confluency. Cells were co-transfected with Na_V plasmid (6-well: 2–4 μg; 24-well: 500–700 ng) and enhanced green fluorescent protein (GFP) plasmid (6-well: 0.8–1.2 μg; 24-well: 80–100 ng) using Lipofectamine^™ LTX with PLUS^™ Reagent (Thermo Fisher) according to the manufacturer’s instructions. GFP was omitted from the transfection of hNa_V1.1 as the construct contains IRES-mScarlet. Following transfection, cells were maintained at 37 °C with 5% CO₂ for 14–48 hours. Transfected cells were washed with PBS, dissociated using trypsin-EDTA, diluted with complete media, and then plated onto 5 mm No. 1 cover glass slips (Warner Instruments, Holliston, MA) 1–2 hours prior to whole-cell patch clamp electrophysiology.

Rat embryonic day 18 hippocampal neurons

Prior to dissection, 5 mm diameter, 0.15 mm-thick round glass coverslips (Warner Instruments) were coated overnight with 1 mg/mL poly-D-lysine hydrobromide (PDL, molecular weight 70,000–150,000 Da, Millipore Sigma) in 0.1 M, pH 8.5 borate buffer in a 37 °C, 5% carbon dioxide, 96% relative humidity incubator.

Pregnant (E17–18) female Sprague Dawley (Strain #001) rats were purchased from Charles River Laboratories and euthanized by carbon dioxide inhalation with subsequent cervical dislocation. All rat euthanasia and dissection procedures were performed in accordance with NIH guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committees (IACUC) of Stanford University. Pregnant dams were singly housed and euthanized on the same day as arrival. Embryonic day 17–18 fetuses of both sexes were surgically removed and hippocampi were dissected into ice-cold Hibernate E (BrainBits, LLC, Springfield, IL) as previously described.^47,62 Each embryo was decapitated post-mortem; the skin and skull of each head were then hemisected along the sagittal plane. Tissues were peeled back to reveal the cortices of the brain, which were rapidly transferred to a petri dish containing ice-cold Hibernate E. Hippocampi were snipped from the underside of each cortex with tweezers and transferred to fresh Hibernate E. Following dissection, cells were dissociated in 2 mL of trypsin-EDTA for 15 min at 37 °C. Subsequently, trypsinized cells were quenched with 10 mL of quenching medium (DMEM high glucose (Thermo Fisher) supplemented with 15% fetal bovine serum, 100 U/mL penicillin–streptomycin, and 1 mM MEM sodium pyruvate (Atlanta Biologicals, Flowery Branch, GA)). The tissue was allowed to settle, the supernatant was removed, and the tissue pellet was rinsed three times with 5 mL of quenching medium. Cells were then manually triturated into 2 mL of plating medium (DMEM supplemented with 10% FBS, 50 U/mL penicillin–streptomycin, and 1 mM MEM sodium pyruvate) by pipetting with a fire-polished 9” borosilicate glass Pasteur pipet (Thermo Fisher).

Cells were plated onto PDL-coated 5 mm glass coverslips in 35 mm tissue culture dishes containing 2 mL of plating medium at a density of 200,000 cells/dish (for voltage-clamp experiments) or 500,000 cells/dish (for current-clamp experiments). After 45 min, coverslips were transferred to new tissue culture dishes containing 2 mL of maintenance medium (neurobasal supplemented with 1x B-27 Supplement, 1x Glutamax, and 50 U/mL penicillin–streptomycin (Thermo Fisher)). Cells were fed every 3–4 days by replacing 2/3 of the conditioned medium.

Mouse Brain Slice

All mouse-related procedures and husbandry were performed according to the guidelines of the Institutional Animal Care and Use Committees (IACUC) of Stanford University. C57BL/6J mice (Strain #000664) were obtained from The Jackson Laboratory (Bar Harbor, ME) and group housed on a 12 h light/dark cycle with food and water ad libitum. Acute brain slices were prepared from adult male and female mice between postnatal day P14–21. Mice were anesthetized with isoflurane and transcardially perfused prior to decapitation. Brains were carefully extracted and 250 μm coronal slices were cut using a vibratome (Leica VT1200 S) in ice-cold slice solution containing (in mM): 110 sucrose, 62.5 NaCl, 2.5 KCl, 6 MgCl₂, 1.25 KH₂PO₄, 26 NaHCO₃, 0.5 CaCl₂, 20 D-glucose, pH 7.35–7.40. Slices were incubated in slice solution for 20 min at 35 °C then transferred to a recovery chamber with artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl₂, 1.25 KH₂PO₄, 26 NaHCO₃, 2 CaCl₂, and 20 D-glucose, pH 7.35–7.40. Brain slices were stored in room temperature ACSF and used for recording 1–5 hours later. All solutions were saturated with carbogen.

METHOD DETAILS

Synthesis

All reagents were obtained commercially. Organic solutions were concentrated under reduced pressure (ca. 60 Torr) by rotary evaporation. HPLC-grade CH₃CN was obtained from commercial suppliers and used as is. N,N-Dimethylformamide (DMF) was passed through two columns of activated alumina prior to use.

Semi-preparative high-performance liquid chromatography (HPLC) was performed on a Varian ProStar model 210. High-resolution mass spectra were obtained from the Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University. Samples were analyzed by LC-flow injection ESI/MS on the Waters Acquity H-Class Plus UPLC and Thermo Exploris 240 BioPharma Orbitrap mass spectrometer scanning m/z 100–1000 Da. A carrier solvent of methanol or acetonitrile with 0.1% formic acid at a flow rate of 0.2mL/min was used to transport the injected sample to the source directly, without chromatographic separation.

Saxitoxin derivatives were quantified by ¹H NMR spectroscopy on a Varian Inova 600 MHz NMR instrument using distilled DMF as an internal standard. A relaxation delay (d₁) of 20 s and an acquisition time (at) of 10 s were used for spectral acquisition. The concentration of the toxin derivative was determined by integrating ¹H signals corresponding to the toxin and a fixed concentration of the DMF standard.

The synthesis of saxitoxin and derivatives was previously described.^25,42,47 STX-amides can be obtained through modification of a previously described synthesis (J. Am. Chem. Soc. 2008, 130, 12630–12631).⁶³ The desired ligands were purified by reversed-phase HPLC (0–60% CH₃CN in 10 mM aqueous trifluoroacetic acid over 60 min unless otherwise noted). Fractions were collected, frozen, and lyophilized to remove all volatiles. Fractions were quantified by ¹H NMR integration and lyophilized again to yield the desired bis-guanidinium derivative. For ¹H NMR spectra, see Data S2. Characterization data for STX-amides are reported below.

Saxitoxin C13 acetamide (STX-NAc 1)

graphic file with name nihms-2193175-f0009.webp

At a flow rate of 4 mL/min and gradient 0–10% CH₃CN in H₂O/trifluoroacetic acid over 20 min, STX-NAc 1 had retention time of 4.91 min and was isolated as a white powder following lyophilization (0.71 μmol, 48%, ¹H NMR quantitation).

¹H NMR (600 MHz, D₂O) δ 4.73 (s, 1H), 3.83 (t, J = 10.0 Hz, 1H), 3.71–3.66 (m, 1H), 3.66–3.58 (m, 2H), 3.23 (dd, J = 14.0, 4.6 Hz, 1H), 2.47 (dd, J = 14.1, 8.0 Hz, 1H), 2.40–2.33 (m, 1H), 2.03 (s, 3H) ppm

HRMS (ESI⁺) calcd for C₁₁H₁₉N₇O₃ 297.1549 found 298.1621 (MH⁺)

Saxitoxin C13 benzamide (STX-NBz 2)

graphic file with name nihms-2193175-f0010.webp

At a flow rate of 4 mL/min and gradient 0–60% CH₃CN in H₂O/trifluoroacetic acid over 60 min, STX-NBz 2 had retention time of 18.61 min and was isolated as a white powder following lyophilization (0.57 μmol, 36%, ¹H NMR quantitation).

¹H NMR (600 MHz, D₂O) δ 7.79 (d, J = 7.7 Hz, 2H), 7.67 (t, J = 7.5 Hz, 1H), 7.57 (dd, J = 7.7, 7.5 Hz, 2H), 3.88–3.81 (m, 2H), 3.78 (t, J = 9.9 Hz, 1H), 3.56 (dt, J = 9.3, 8.8 Hz, 1H), 3.40 (dt, J = 9.3, 8.4 Hz, 1H), 2.46–2.40 (m, 1H), 2.39–2.32 (m, 1H) ppm

Note: *H5 obscured by HDO peak

HRMS (ESI⁺) calcd for C₁₆H₂₁N₇O₃ 359.1706 found 360.1779 (MH⁺)

Saxitoxin C13 para-toluamide (STX-N^pTl 3)

graphic file with name nihms-2193175-f0011.webp

At a flow rate of 4 mL/min and gradient 0–60% CH₃CN in H₂O/trifluoroacetic acid over 60 min, STX-N^pTl 3 had retention time of 17.51 min and was isolated as a white powder following lyophilization (0.21 μmol, 13%, ¹H NMR quantitation).

¹H NMR (600 MHz, D₂O) δ 7.64 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 3.82–3.70 (m, 3H), 3.51 (dd, J = 12.9, 3.3 Hz, 1H), 3.34 (dt, J = 9.3, 9.3 Hz, 1H), 2.40–2.35 (m, 4H), 2.34–2.27 (m, 1H) ppm

Note: *H5 obscured by HDO peak

HRMS (ESI⁺) calcd for C₁₇H₂₃N₇O₃ 373.1862 found 374.1942 (MH⁺)

Saxitoxin C13 meta-toluamide (STX-N^mTl 4)

graphic file with name nihms-2193175-f0012.webp

At a flow rate of 4 mL/min and gradient 0–60% CH₃CN in H₂O/trifluoroacetic acid over 60 min, STX-N^mTl 4 had retention time of 17.56 min and was isolated as a white powder following lyophilization (0.20 μmol, 14%, ¹H NMR quantitation).

¹H NMR (600 MHz, D₂O) δ 7.62 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 4.84 (s, 1H), 3.87–3.81 (m, 2H), 3.78 (t, J = 10.1 Hz, 1H), 3.60–3.52 (m, 1H), 3.41 (dt, J = 9.9, 8.1 Hz, 1H), 2.46–2.40 (m, 4H), 2.36 (dt, J = 13.9, 9.9 Hz, 1H) ppm

HRMS (ESI⁺) calcd for C₁₇H₂₃N₇O₃ 373.1862 found 374.1942 (MH⁺)

Saxitoxin C13 ortho-toluamide (STX-N°Tl 5)

graphic file with name nihms-2193175-f0013.webp

At a flow rate of 4 mL/min and gradient 0–50% CH₃CN in H₂O/heptafluorobutyric acid over 50 min, STX-N°Tl 5 had retention time of 35.49 min.

¹H NMR (600 MHz, D₂O) δ 7.47 (dd, J = 7.7, 7.6 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.34 (dd, J = 7.7, 7.6 Hz, 1H), 4.84 (s, 1H), 3.88–3.79 (m, 3H), 3.56–3.46 (m, 2H), 2.47–2.41 (m, 1H), 2.41–2.33 (m, 4H) ppm

HRMS (ESI⁺) calcd for C₁₇H₂₃N₇O₃ 373.1862 found 374.1934 (MH⁺)

Saxitoxin C13 cyclohexylamide (STX-NCy 6)

graphic file with name nihms-2193175-f0014.webp

At a flow rate of 4 mL/min and gradient 0–60% CH₃CN in H₂O/trifluoroacetic acid over 60 min, STX-NCy 6 had retention time of 16.60 min and was isolated as a white powder following lyophilization (0.56 μmol, 36%, ¹H NMR quantitation).

¹H NMR (600 MHz, D₂O) δ 4.73 (s, 1H), 3.84 (dt, J = 10.2, 1.8 Hz, 1H), 3.7–3.64 (m, 2H), 3.63–3.57 (m, 1H), 3.23 (dt, J = 9.9, 9.2 Hz, 1H), 2.50–2.44 (m, 1H), 2.37 (dt, J = 14.0, 9.9 Hz, 1H), 2.29–2.21 (m, 1H), 1.84–1.75 (m, 4H), 1.68 (d, J = 12.8 Hz, 1H), 1.40–1.25 (m, 4H), 1.25–1.16 (m, 1H) ppm

HRMS (ESI⁺) calcd for C₁₆H₂₇N₇O₃ 365.2175 found 366.2246 (MH⁺)

Plasmids

A human Na_V1.1 (hNa_V1.1) construct, pIR-CMV-SCN1A-Variant-1-IRES-mScarlet, was obtained as a gift from Dr. Al George (Northwestern University Feinberg School of Medicine, Department of Pharmacology) (Addgene plasmid #162278; http://n2t.net/addgene:162278; RRID:Addgene_162278).⁶⁴ CMV-promoted FLAG-rNa_V1.2, rNa_V1.3, and myc-mouse Na_V1.6 (myc-mNa_V1.6) in modified pcDNA3.1(+) vectors were constructed by VectorBuilder (Chicago, IL) or GenScript (Piscataway, NJ). The full-length cDNA used for excision and insertion into low-copy modified pcDNA3.1(+) were from oocyte expression vectors provided by Dr. A. L. Goldin (University of California, Irvine, Department of Microbiology & Molecular Genetics).^47,65 MT1-promoted rNa_V1.4 in a pZem288 vector was provided by Dr. S. Rock Levinson (University of Colorado, School of Medicine, Department of Physiology and Biophysics).⁶⁶ CMV-promoted hNa_V1.5 in the mammalian expression vector pcDNA3.1(+) was gifted by Dr. T. R. Cummins (Indiana University, Department of Biology). CMV-promoted rNa_V1.7 (Accession No. NM_133289.1) in a pcDNA3.1(+) vector was purchased from GenScript. All plasmids were amplified in DH5α (Thermo Fisher, Waltham, MA) or stbl2 (Thermo Fisher) competent cells grown at 37 °C and purified using the QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. Due to the inherent instability of the plasmid, the rNa_V1.7 wild-type construct and mutant channels were prepared in CopyCutter EPI400 Competent Cells (Biosearch Technologies, Hoddesdon, United Kingdom) at 30 °C.

Mutagenesis

Na_V mutants were prepared by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. Primers (Table S1) were synthesized by Elim Biopharmaceuticals, Inc. (Hayward, CA). Product plasmids were amplified and purified as described above. Following purification, mutations were confirmed through DNA sequencing with Elim Biopharmaceuticals, Inc. or Plasmidsaurus (South San Francisco, CA). Double mutant channels rNa_V1.3 D1372L/G1664A and rNa_V1.7 D1410L/G1702A were prepared by GenScript and amplified and purified as described previously. Several rNa_V1.4 mutants were previously reported:

Y401S, E403D, W756A, I757A, E758D, M1240I, M1240A, M1240N, D1241T, D1241E, D1241N, D1241A, D1532E^25,67
M1240T, D1241I⁶⁸
D762E, G1533A, E403Q⁴²
D1532N^39,40
Y401C^39,69

Sodium current recordings

All voltage-clamp recordings were acquired at 100 kHz with an Axopatch 200B amplifier and digitized with a Digidata 1322A (Molecular Devices, San Jose, CA). Signals were filtered with a built-in lowpass, four-pole Bessel filter with a cutoff of 5 kHz. For CHO-K1 cells, fire-polished, thin-walled borosilicate pipettes (1.0–5.0 MΩ) contained an internal solution composed of 40 mM NaF, 1 mM EDTA, 20 mM HEPES, and 125 mM CsCl (pH 7.4 with 50 wt. % aqueous CsOH, 330–335 mOsm). The external solution comprised 160 mM NaCl, 2 mM CaCl₂, and 20 mM HEPES (pH 7.4 with 50 wt. % aqueous CsOH, 325–330 mOsm).

Voltage-clamp recordings in cultured hippocampal neurons were performed at DIV 6–8 using fire-polished, thin-walled borosilicate pipettes (3–10 MΩ) with an internal solution composed of 114.5 mM gluconic acid, 114.5 mM CsOH, 2 mM NaCl, 8 mM CsCl, 10 mM MOPS, 4 mM EGTA, 4 mM MgATP, and 0.3 mM Na₂GTP (pH 7.3 with CsOH, 260–265 mOsm). The external solution was Hibernate E low fluorescence (BrainBits, Springfield, IL) (260–275 mOsm with NaCl).

The outer membrane was kept at a holding potential of −100 mV for CHO-K1 cells and −80 mV for neurons unless otherwise noted. Prior to recording, cells were equilibrated for >5 min after establishing whole-cell configuration. Series resistance was compensated at 80–95% with a τ_lag of 20–30 or 35 ms for CHO-K1 cells or hippocampal neurons, respectively. Leak currents were subtracted using a standard P/4 protocol of opposite polarity. All measurements were recorded at room temperature (20–25 °C). Data were collected on cells exhibiting >0.75 nA peak current upon depolarization (unless otherwise noted), leak current <0.2 nA, and I/I₀ <15% of I₀ upon STX or STX-amide wash-out. Due to low current density in both WT and mutant rNa_V1.3 channels, data were collected on cells exhibiting >0.5 nA of current.

Screening and Concentration-response Curves

Currents were elicited by 10 ms step depolarizations from a holding potential (−100 mV for CHO-K1 cells expressing hNa_V1.1, rNa_V1.2, rNa_V1.3, rNa_V1.4, mNa_V1.6, or rNa_V1.7; −120 mV for CHO-K1 cells expressing hNa_V1.5; or −80 mV for E18 hippocampal neurons) to 0 mV at a rate of 0.5 Hz. Toxin solutions were perfused into the bath at a rate of 1 mL/min in the order of lowest to highest concentration through manual perfusion. Cells were bathed in each toxin concentration for 60 seconds with continuous recording to allow complete wash on and equilibration. Cells were perfused with ~2 mL of external solution following the acquisition of electrophysiology data for three different ligand concentrations. After recording data for the final ligand concentration, application of external solution was performed to ensure complete ligand wash-out and restoration of peak current to within 15% of I₀.

Peak sodium currents were normalized to initial peak currents (I₀), plotted against toxin concentration, and analyzed using Prism 10.5 (GraphPad Software, LLC, San Diego, CA). For screening results, percent reduction in initial current is plotted. For concentration-response curves, normalized sodium current data were fit to a dose-response curve with a nonlinear, least squares regression with a four-parameter variable slope inhibitor versus response equation in GraphPad Prism, as follows:

Y = \frac{1}{1 + 10^{(l o g I C_{50} - x) HillSlope}}

where $Y$ is the normalized sodium current and x is the concentration of toxin. The bottom and top parameters were constrained to 0 and 1 respectively. The Hill Slope and logIC₅₀ were set to variable. The number of observations (n) was ≥3 for all reported data. Errors in IC₅₀ values and Hill coefficient values are expressed as s.e.m.

Kinetic Measurements of Block

Currents were elicited by 10 ms step depolarizations from −100 mV to 0 mV at a rate of 0.5 Hz. Using a pinch valve perfusion system (Automate Scientific, Berkeley, CA) to control the perfusion rate at 1.5 mL/min, 1.5 mL of STX or STX-N^mTl 4 was washed onto cells and equilibrated for 1 min. Cells were then washed with 1–2 mL of external solution until full return of current. Only cells that returned within 10% of initial peak current were used in the analysis. A ligand concentration that approximated the respective IC₅₀ value was used to measure the rate of binding (5 nM STX in WT, 4.5 μM 4 in WT, and 25 nM 4 in rNa_V1.4 D1241L/G1533A). Peak current was normalized to the initial current and plotted against time. The decay in current during wash on and recovery of current during wash out were fit to exponential curves to obtain the on-rate time constant (τ_on) and off-rate time constant (τ_off). Rate constants for block (k_on) and recovery (k_off) were derived according to the equations by Hahin and Strichartz, as follows:⁵⁹

k_{o n} = \frac{\frac{1}{τ_{o n}} - \frac{1}{τ_{o f f}}}{[toxin]}

k_{o f f} = \frac{1}{τ_{o f f}}

State-dependence of Binding

Currents were elicited by a 10 ms test pulse to 0 mV at a frequency of 0.5 Hz. Test pulses were preceded by 200 ms conditioning pulses to −70 mV or −120 mV to sample different conformational states. A baseline involving 5 scans was recorded prior to toxin exposure. Using a perfusion system (Automate Scientific, Berkeley, CA), 1.5 mL of STX or STX-N^mTl 4 was washed onto cells at a rate of 1.5 mL/min and allowed to equilibrate for 1 min. Cells were then washed with 1–2 mL of external solution. Only cells that returned within 10% of initial peak current were used for analysis. The baseline and ligand-treated peak current were recorded as the average of 5 scans.

Voltage-dependence of Conductance (Activation)

Currents were evoked using the stimulus protocol depicted in Figure S4A at sampling rate of 50 kHz. Peak current was converted to conductance (G) using the equation I_Na/(V_m–V_rev), where I_Na is the measured peak sodium current, V_m is the applied membrane potential, and V_rev is the experimentally determined reversal potential for sodium. Conductance at each potential step was normalized to the maximum conductance and plotted against voltage. The G–V curve was fit to a sigmoidal curve constrained to [0,1] with variable slope and the midpoint of the curve, V_1/2act, was calculated in GraphPad Prism. Cells with >0.5 nA current and experimentally determined V_rev between 32–38 mV, within 3 mV of the calculated V_rev, were used for analysis.

Voltage-dependence of Availability (Steady-state Inactivation)

Currents were evoked using the stimulus protocol depicted in Figure S4C at a sampling rate of 100 kHz. Peak current was normalized to the largest current across sweeps and plotted against voltage. The I–V curve was fit to a sigmoidal curve constrained to [0,1] with variable slope and the midpoint of the curve, V_1/2inact, was calculated in GraphPad Prism. Cells with >0.5 nA current were used for analysis.

Ion Selectivity

The internal solution was composed of 40 mM CsF, 1 mM EGTA, 20 mM HEPES, and 125 mM CsCl (pH 7.4 with 50 wt. % aqueous CsOH, 325 mOsm); the external solution comprised 160 mM X-Cl (X = Na, K) or 110 mM XCl₂ (X = Ca), 2 mM CaCl₂, and 20 mM HEPES (pH 7.4 with 50 wt. % aqueous CsOH, 325–330 mOsm). Liquid junction potentials (LJP) were zeroed at the start of each experiment in Na⁺ external. LJPs were calculated and experimental confirmed to be −1.6 mV for K⁺ and −10.5 mV for Ca²⁺. The LJP was corrected during data analysis.

Cells were first patched in Na⁺ external solution and equilibrated for at least 5 min after establishing the whole-cell and voltage-clamp configuration. Currents were evoked using the protocol depicted in Figure S5B at a sampling rate of 50 kHz. External solution was replaced through perfusion to either K⁺ or Ca²⁺ external solution, and currents were evoked by the same protocol.

To separate sodium channel currents from leak currents, whole-cell currents were measured as the difference between peak currents in a 13.5–20 ms window and average sustained current was measured between 150–160 ms. Data were filtered during analysis by a 2 kHz low-pass Bessel filter applied in Clampfit. The peak current at each voltage step was normalized to the largest peak current in Na⁺ external conditions.

Action potential recordings

Cultured hippocampal neurons (DIV 9–13)

Current clamp recordings were acquired at 100 kHz and filtered with a built-in lowpass, four-pole Bessel filter with a cutoff of 10 KHz. The internal solution comprised 105 mM potassium gluconate, 30 mM KCl, 10 mM HEPES, 10 mM Na₂phosphocreatine, 1 mM EGTA, 4 mM MgATP, and 0.3 mM Na₂GTP (pH 7.4 with KOH, 275–280 mOsm); the external solution comprised Hibernate E low fluorescence adjusted to 285–300 mOsm with NaCl. To ensure cells maintained a resting membrane potential between −50 and −70 mV that did not vary more than ±5 mV over the course of the experiment, cells were injected with a holding current between 0 and −100 pA. Series resistance was compensated at 90–95% with a τ_lag of 35 ms.

Single AP Recordings

Single action potentials were elicited by a 25 ms current injection to +100 pA at a rate of 0.2 Hz. A 200 ms injection of −30 pA was included after the elicited AP to test input resistance. Five traces were recorded at baseline, with STX-N^mTl 4 applied, and following wash-out of 4. Amplitude, full width at half maximum, and maximum dV/dt were calculated in Clampfit for each trace, and values were normalized to the average baseline values. Cells were excluded if, after wash-out, both amplitude and full width at half maximum exceeded ±15% of the baseline value.

AP Trains

Action potentials were elicited by a 750 ms current injection of +50–100 pA at a rate of 0.125 Hz. A 500 ms injection to −30 pA was included prior to the elicited AP trains to test input resistance. Cells were selected for analysis based on firing a minimum of six APs per 750 ms. The number of APs were counted in five traces before application of STX-N^mTl 4, after application, and post wash-out. The average number of APs recorded under each condition were normalized to the average number of APs in the baseline. Cells were excluded if, after wash-out, the number of APs fired exceeded ±15% of the baseline value.

Acute brain slices

Individual brain slices were placed in an RC-27 recording chamber (Warner Instruments) and superfused with carbogen-saturated ACSF (33–36 °C) at a flowrate of 2–3 mL/min. Layer 5 pyramidal neurons in the primary motor cortex (M1) were visualized with a BX51WI upright microscope (Olympus, Center Valley, PA) equipped with IR-DIC optics and a 40X 0.8 NA water immersion objective (LUMPLFLN, Olympus). Whole-cell recordings were made using borosilicate pipettes (4–7 MΩ) filled with internal solution containing (in mM): 130 K-gluconate, 10 KCl, 10 Na-phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP, 10 HEPES, 290 mOsm, pH 7.2 adjusted with KOH. STX-N^mTl 4 was dissolved in water and used for recording within 24 hours. To determine the acute effects of STX-N^mTl 4 on the somatic intrinsic excitability in M1 layer 5 pyramidal neurons, continuous AP trains were evoked using 1s current injections ranging from 100–400 pA during 5 min of baseline (ACSF), during 10 min of STX-N^mTl 4 perfusion, and 10 min of wash-out (ACSF). Resting membrane potential at the start of the recordings was between −60 and −80 mV. A small amount of holding current (±150 pA) was injected to bring each cell close to −70 mV prior to AP recordings. Input resistance was monitored throughout the duration of all recordings using a −50 pA hyperpolarizing pulse. Recordings were not included in the final analysis if input resistance changed by >20%. Pipette capacitance neutralization and bridge balance were manually adjusted prior to current-clamp recordings. The liquid junction potential was not corrected. Current-clamp recordings in slice were sampled at 20 kHz and filtered at 10 kHz using a MultiClamp 700B and Digidata 1550B and analyzed using pCLAMP11 (Molecular Devices). Data are presented as mean value ± s.e.m., where n or N represent number of cells or animals, respectively.

Computational Modeling (Data S1)

In silico docking experiments for STX-N^mTl 4 were performed using the Maestro v14.3 environment (Schrödinger) with the Glide Docking model. The ligand was prepared using the LigPrep module and docking experiments were performed using the Glide docking package with an OPLS4 force field.^70,71 Docking was performed with PDB 6J8G (hNa_V1.7 with STX bound) using a 20 Å receptor grid centered on STX in Site 1.⁶¹ At least 10 docking poses were generated for all docking experiments. The top pose generated from docking was exported to PyMOL v3.1 and modeled with PDB 6J8G.

Ethics oversight

All procedures complied with animal care standards set forth by the National Institute of Health and were approved by the Stanford Institutional Animal Care & Use Committee (IACUC).

QUANTIFICATION AND STATISTICAL ANALYSIS

All data analysis was performed using the software packages pCLAMP Clampfit v11 (Molecular Devices), Microsoft Excel, or Prism v10.5. The statistical significance of differences between mean values was evaluated using Student’s t-test, paired t-test, or one-way ANOVA with Dunnett’s correction with p < 0.05 considered significant. Results are presented as mean ± s.e.m. unless indicated otherwise. P-values are denoted as ns (not significant), p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001. Data are typically presented as mean value ± s.e.m., where n or N represent number of cells or animals, respectively. All statistical details of experiments can be found in the figure legends or methods.

Supplementary Material

Data S2. ¹H NMR spectra, related to STAR Methods

NIHMS2193175-supplement-1.pdf^{(278.1KB, pdf)}

Document S1. Figures S1–S9, Protocols S1, and Supplemental References

NIHMS2193175-supplement-2.pdf^{(1.9MB, pdf)}

Data S1. PyMOL Session File of PDB 6J8G with STX and STX-N^mTl, Related to Figure 3

NIHMS2193175-supplement-3.pse^{(3.3MB, pse)}

Table S1. Primers for mutagenesis, related to STAR Methods

NIHMS2193175-supplement-4.xlsx^{(15KB, xlsx)}

Key resources table.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
MAX Efficiency^™ DH5α Competent Cells	Thermo Fisher	Cat#18258012
MAX Efficiency^™ Stbl2^™ Competent Cells	Thermo Fisher	Cat#10268019
CopyCutter EPI400 Chemically Competent Cells	Biosearch Technologies	Cat#C400CH10
Chemicals, peptides, and recombinant proteins
Saxitoxin	Du Bois Laboratory, Stanford University	N/A
STX-N^mTl and other derivatives	Du Bois Laboratory, Stanford University	N/A
Lipofectamine^™ LTX with PLUS^™ Reagent	Thermo Fisher	Cat#15338100
QuikChange Lightning Site-Directed Mutagenesis Kit	Agilent	Cat#210519
Experimental models: Cell lines
Hamster: Chinese Hamster Ovary (CHO)-K1 Cells	ATCC	CCL-61^TM
Experimental models: Organisms/strains
Rat: CR^® (Sprague Dawley)	Charles River Laboratories	Strain #:001; RRID: RGD_734476
Mouse: C57BL/6J	Jackson Laboratory	Strain #:000664; RRID:IMSR_JAX:000664
Oligonucleotides
Primers for Mutagenesis, see Table S1	This Paper	N/A
Recombinant DNA
pIR-CMV-SCN1A-Variant-1-IRES-mScarlet	DeKeyser, et al.⁶⁴	Addgene #162278 RRID:Addgene_162278
pcDNA3.1-CMV-FLAG-rNaV1.2	VectorBuilder; backbone modified from plasmid received from Dr. A. L. Goldin, University of California, Irvine	N/A
pcDNA3.1-CMV-rNaV1.3	VectorBuilder; backbone modified from plasmid received from Dr. A. L. Goldin, University of California, Irvine	N/A
pZem288-MT1-rNaV1.4	Dr. S. Rock Levinson, University of Colorado, School of Medicine	N/A
pcDNA3.1-CMV-hNaV1.5	Dr. T. R. Cummins, Indiana University	N/A
pcDNA3.1-CMV-myc-mNav1.6	GenScript; backbone modified from plasmid received from Dr. A. L. Goldin, University of California, Irvine	N/A
pcDNA3.1-CMV-rNav1.7	GenScript	N/A
Software and algorithms
pCLAMP v11	Molecular Devices	RRID: SCR_011323 https://support.moleculardevices.com/s/article/AxonpCLAMP-11-Electrophysiology-Data-Acquisition-Analysis-Software-Download-Page
GraphPad Prism v10.5	GraphPad	RRID: SCR_002798 https://www.graphpad.com/features
Chemdraw v25.5	Revvity	RRID: SCR_016768 https://revvitysignals.com/products/research/chemdraw
Maestro v14.3	Schrödinger	RRID: SCR_016748 https://www.schrodinger.com/maestro
PyMOL v3.1	Schrödinger	RRID: SCR_000305 https://www.pymol.org/

Open in a new tab

HIGHLIGHTS.

A designer saxitoxin targets a mutant Na_V with ca. 100:1 selectivity over WT channels
Block of ion conduction in mutant channels occurs rapidly (seconds) and reversibly
APs in native neurons and CNS tissue are unaffected at operative ligand concentrations
Applicable for studies of any one of multiple Na_V subtypes, Na_V1.1–1.4, 1.6, 1.7

SIGNIFICANCE.

Neuronal excitability relies on the tightly regulated expression and discrete subcellular localization of voltage-gated sodium channels (Na_Vs), large membrane protein complexes responsible for initiating and propagating action potentials. Channel malfunction underlies multiple human pathologies (e.g., epilepsy, pain, arrhythmia, and musculoskeletal disorders), clinical findings that drive considerable interest in Na_Vs as drug targets and that motivate research to better understand channel physiology. To investigate Na_V function in health and disease, pharmacological tools that enable rapid and reversible manipulation of individual Na_V isoforms are needed along with methods for differentiating between the same Na_V isoform present in two or more cell types (e.g., Na_V1.6 in neurons vs glia). The latter is currently not possible even with the availability of potent, subtype-selective Na_V ligands. Accordingly, we have capitalized on chemical design and protein engineering to address these challenges by developing a general technology for interrogating Na_V1.1, 1.2, 1.3, 1.4, 1.6, or 1.7 physiology. A synthetic derivative of the bis-guanidinium toxin, saxitoxin (STX), is paired with two discrete outer pore-forming amino acid mutations to achieve ca. 100:1 selectivity for the engineered channel over all wild-type isoforms. The designer ligand is nanomolar potent against the mutant channel and acts within seconds to block sodium ion conduction; washout occurs rapidly and completely to restore channel function. The advent of a unique pharmacological inhibitor of a genetically encoded Na_V subtype will unlock new studies of Na_V physiology and afford precise control of action potential signaling given the requisite role of Na_Vs in electrogenesis.

ACKNOWLEDGMENTS

The authors thank Roshni Bhaskar for her assistance with initial studies to prepare STX-amide ligands, Dr. Erica Liu and Prof. Bianxiao Cui for help with neuron preparations, and Prof. Christopher Makinson, Dr. Torben Neelands, and Steven Miller for helpful discussions. This work was supported by National Institutes of Health (NIH) R01-GM117263. E.R.P. was supported by the Stanford ChEM-H Chemistry/Biology Interface Program, the National Institute of General Medical Sciences (NIGMS) of the NIH under Award Number T32-GM120007, and the National Sciences Foundation (NSF) Graduate Research Fellowship Program. H.S.H. was supported by the Department of Defense (DOD) National Defense Science and Engineering Graduate (NDSEG) Fellowship. This work utilized a Thermo Exploris 240 LC/MS system (RRID:SCR_022216) that was purchased with funding from Stanford C-ShaRP (RRID:SCR_022986) and was supported by Stanford University Mass Spectrometry (RRID:SCR_017801).

Footnotes

DECLARATION OF INTERESTS

J.D. is a cofounder and holds equity shares in TI Therapeutics, Inc., a start-up company interested in developing subtype-selective modulators of sodium channels.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S2. ¹H NMR spectra, related to STAR Methods

NIHMS2193175-supplement-1.pdf^{(278.1KB, pdf)}

Document S1. Figures S1–S9, Protocols S1, and Supplemental References

NIHMS2193175-supplement-2.pdf^{(1.9MB, pdf)}

Data S1. PyMOL Session File of PDB 6J8G with STX and STX-N^mTl, Related to Figure 3

NIHMS2193175-supplement-3.pse^{(3.3MB, pse)}

Table S1. Primers for mutagenesis, related to STAR Methods

NIHMS2193175-supplement-4.xlsx^{(15KB, xlsx)}

Data Availability Statement

Data: All data reported in this paper will be shared by the lead contact upon request.
Code: This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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