Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain


Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

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Figure 1: Hm1a selectively targets Nav1.1 in sensory neurons.
Figure 2: Hm1a targets S3b–S4 and S1–S2 loops in DIV to inhibit fast inactivation.
Figure 3: Nav1.1 is expressed by myelinated, non-C-fibre neurons in sensory ganglia.
Figure 4: Hm1a elicits non-inflammatory pain and bilateral mechanical allodynia.
Figure 5: Colonic afferents display increased sensitivity to Hm1a in a model of IBS.

Accession codes

Data deposits

Sequences and activity profiles for Hm1a and Hm1b can be found in the ArachnoServer database49 under accession numbers AS000224 and AS002363, respectively.


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We thank the Deutsche Arachnologische Gesellschaft and particularly I. Wendt, J. Broghammer, A. Schlosser, B. Rast, M. Luescher, C. and F. Schneider and H. Auer for providing arthropods for milking; W. Catterall for providing floxed Nav1.1 mice; and J. Poblete, J. Maddern, T. O’Donnell and A. Harrington for technical assistance. This work was supported by a T32 Postdoctoral Training Grant from the UCSF CVRI (J.D.O.), Ruth Kirschstein NIH postdoctoral (F32NS081907 to J.D.O.) and predoctoral (F31NS084646 to J.G. and F30DE023476 to J.J.E.) fellowships, the National Institutes of Health (R37NS065071 and R01NS081115 to D.J., R01NS091352 to F.B., R01NS040538 and R01NS070711 to C.L.S., and R37NS014627 and R01DA29204 to A.I.B.), the National Health and Medical Research Council of Australia (Project Grant APP1083480 to S.M.B., Program Grant APP1072113 and Principal Research Fellowship APP1044414 to G.F.K.), and a grant from the Wellcome Trust to A.I.B. S.M.B. is a NHMRC R.D Wright Biomedical Research Fellow.

Author information




J.D.O., V.H., E.A.B.U., G.F.K. and D.J. carried out venom collection and screening, toxin purification and characterization. Z.D. and P.A. carried out Hm1a synthesis. J.D.O., J.G., C.Z., D.J. and F.B. designed, performed, and analysed electrophysiological and calcium imaging experiments to determine toxin mechanism and selectivity. J.J.E., J.D.O., X.W. A.I.B, and D.J. designed, performed and analysed histological experiments. A.D.W. and C.L.S. designed, performed, and analysed skin-nerve recordings. X.W., J.D.O., D.J., and A.I.B. designed, performed, and analysed behavioural experiments to assess somatic function. J.C., S.G.-C., L.G., G.Y.R. and S.M.B. designed, performed and analysed studies relating to colonic afferent and patch clamp pharmacological studies. All authors contributed to the discussion and interpretation of the results. J.D.O. and D.J. wrote the manuscript with contributions and suggestions from all authors.

Corresponding authors

Correspondence to Frank Bosmans or Glenn F. King or David Julius.

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Competing interests

The authors’ institutions have submitted provisional patent applications based, in part, on the work described in this article.

Extended data figures and tables

Extended Data Figure 1 Hm1a and Hm1b selectively target Nav1.1 in sensory neurons.

a, Left, HPLC chromatogram showing reversed-phase C18 fractionation of Heteroscodra maculata venom; peaks containing Hm1a and Hm1b are labelled. Peptide sequences as determined by Edman degradation are displayed above. Middle, MALDI–TOF spectra of native undigested Hm1b (top) and native Hm1b digested with carboxypeptidase Y for 20 min (bottom), with inserted spectra showing the monoisotopic mass of each in daltons (Da). The observed mass difference of 146 Da between the intact and digested Hm1b corresponds to the final residue, Phe, with an amidated C terminus. Right, chromatograms show reversed-phase C18 HPLC profiles of native and synthetic Hm1a, which were indistinguishable when co-injected. b, Representative currents from oocytes expressing hNav subtypes before (black) and after (grey) bath application of ICA-121431 (500 nM). Currents were elicited during 1-Hz stimulation to induce use-dependent block. c, Top, amino acid sequence comparison of Hm1a with SGTx1, a related, but non-selective fast-inactivation inhibitor. Bottom, representative calcium imaging experiment comparing ICA-121431-mediated block of Hm1a- or SGTx1-evoked responses in cultured embryonic DRG neurons, with group data at right (**P < 0.01, ***P < 0.001, n = 4). d, Top, fraction of P0 mouse neurons responding to Hm1a versus SGTx1 (**P < 0.01). Bottom, ratiometric calcium responses elicited by SGTx1 (500 nM) in the presence and absence of ICA-121431 (500 nM). e, Dose–response curves for Hm1a inhibition of fast inactivation in oocytes expressing Nav1.1, Nav1.2 or Nav1.3. Sustained current at the end of a 100-ms pulse is normalized to peak current to quantify magnitude of the effect. EC50 values for hNav1.1 = 38 nM, hNav1.2 = 236 nM and hNav1.3 = 220 nM. f, Representative traces from oocytes expressing hNav subtypes in response to a saturating dose (on hNav1.1) of purified Hm1b during a 100-ms depolarization. g, rKv2.1 chimaeras containing different Nav1.9 S3b–S4 motifs were tested for sensitivity to hHm1a (100 nM). Representative traces (top) and summary data (bottom) show a lack of toxin sensitivity for each chimaera. h, Top left, representative currents from oocytes expressing mKv4.1 before (black) and after (red) bath application of Hm1a (5 μM). Middle, quantification of mKv4.1 blockade by synthetic or native Hm1a. Top right, comparison of sustained current during application of native or synthetic Hm1a (1 μM) shows similar effects on Nav1.1 inactivation. Bottom, representative traces (left) showing that outward currents in P0 trigeminal mouse neurons are unaffected by Hm1a (500 nM). Scatter plot (right, n = 10) shows no significant difference. i, Percentage of Hm1a (500 nM)-responsive neurons in various culture conditions as assessed by calcium imaging (n = 3–4,*P < 0.05). Error bars represent mean ± s.e.m. P values based on two-way ANOVA with post hoc Tukey’s test (c) or unpaired two-tailed Student’s t-test (d, i).

Extended Data Figure 2 Hm1a selectivity depends on DIV S1–S2 and S3b–S4 regions.

a, Top, alignments between Kv2.1 and hNav1.1 S3b–S4 regions from each domain (as indicated) with sequence of chimaeras shown below each alignment. Bottom, GV relationships from chimaeric channels expressed in oocytes in the absence (black) and presence (colours) of Hm1a (100 nM). b, Sequence alignment of hNav1.1 and rNav1.4 showing putative transmembrane segments (green) and regions swapped in chimaeric channels (grey). c, Top, using the background of Nav1.4 chimaera containing the S3b–S4 and S5–S6 regions of Nav1.1, individual residues were mutated in the S1–S2 loop to the cognate residue in Nav1.1. The D1376T and Y1379S point mutants in the chimaeric rNav1.4 channel reveal an increase in peak current after 100 nM Hm1a application (red) relative to untreated controls (black). Filled circles denote GV relationships, where oocytes were depolarized for 50 ms in 5-mV steps from a holding potential of −90 mV. Open circles denote steady-state inactivation (I/Imax) relationships, where oocytes were depolarized from −90 mV to +5 mV in 5-mV increments for 50 ms preceeding a 50-ms step to −15 mV. Middle, dot plot detailing per cent increase in peak conductance of each point mutant in response to 100 nM Hm1a treatment. Each point represents a single oocyte; red bars indicate 95% confidence interval. Mutations highlighted in orange (D1376T and Y1379S) are statistically different from S3b–S4/S5–S6 control (*P < 0.01, Student’s t-test). The Q1372E mutant did not generate currents. Bottom, alignment of DIV S1–S4 regions from relevant mouse and human Nav isoforms. Orange highlights location of residues in the S1–S2 loop that putatively contribute to the toxin effect. d, Left, stylized DIV with transmembrane segments represented as circles and extracellular loops as bars (black for native rNav1.4 channel and green for portions transplanted from hNav1.1). Middle, traces displaying effect of Hm1a on each chimaera depolarized to −15 mV from a holding potential of −90 mV. Right, conductance–voltage (G/Gmax) and steady-state inactivation (I/Imax) relationships of each channel and chimaera before and after toxin (black and red, respectively) across a voltage range spanning −90 mV to 0 mV from a holding potential of −90 mV in 5-mV increments. Scale bars as in Fig. 2. e, Dot plots displaying the effect of 100 nM Hm1a on peak current (left) and persistent current (right). Data in the left plot were generated by dividing peak conductance after Hm1a application by the peak conductance before. Right plot shows persistent current divided by peak current before (black) or after (red) toxin addition. Persistent current was determined by averaging current from the final millisecond of depolarization to 0 mV from a holding potential of −90 mV. Vertical bars indicate 95% confidence interval.

Extended Data Figure 3 Nav1.1 is expressed by myelinated, non-C-fibre sensory neurons.

a, Representative images showing expression of various molecular markers and their overlap with Nav1.1 transcripts. Markers include immunohistochemical staining for CGRP and tyrosine hydroxylase (TH) and in situ histochemistry for TRPM8 and 5-HT3 ion channel transcripts. Quantification of overlap for these markers is shown in Fig. 3. b, Quantification of the number of toxin-responsive cells in P0 mouse trigeminal cultures as assessed by calcium imaging (leftmost column) and the percentage of toxin-sensitive cells that responded to other agonists (1-(m-chlorophenyl)-biguanide (mCPBG), allyl isothiocyanate (AITC), capsaicin, and menthol activate 5-HT3, TRPA1, TRPV1 and TRPM8 channels, respectively), or bound the lectin IB4. c, Table including conduction velocity and Von Frey thresholds for skin-nerve experiments presented in Fig. 3d. Error bars represent mean ± s.e.m.

Extended Data Figure 4 Control experiments.

These data show control experiments related to Fig. 4. a, Representative DRG sections from peripherin-Cre adult mouse showing neurons that express Cre recombinase as visualized using a floxed-stop YFP reporter mouse. In situ hybridization histochemistry shows overlap with Nav1.1 transcripts (right). b, Quantification of overlap between YFP and Nav1.1. c, Comparison of ATF3 induction in DRG following sciatic nerve ligation (SNI) or intraplantar Hm1a injection. SNI induced ATF3 expression in >50% of DRG neurons whereas ATF3 induction after Hm1a injection was indistinguishable from vehicle (measured 1 or 3 days post-injection). d, Peripherin-Cre × floxed Nav1.1 mice were compared with wild-type littermates in the rotarod test. No significant differences were observed (unpaired Student’s t-test). Error bars represent mean ± s.e.m.

Extended Data Figure 5 A subset of colonic afferents does not express functional Nav1.1.

a, Left, representative ex vivo colonic single fibre recording from an Hm1a (100 nM)-non-responsive high-threshold fibre from a healthy mouse (arrows indicate application and removal of 2 g Von Frey hair stimulus). Middle, group data showing lack of Hm1a-mediated responses from a subset (9 out of 15) of fibres. Right, group data showing a population (5 out of 10) of healthy, high-threshold mechanoreceptor colonic afferents unaltered by ICA-121432 in the presence or absence of Hm1a (100 nM). b, Left, representative whole–cell current clamp recording of a retrogradely traced colonic DRG neuron in response to 500-ms current injection at rheobase. Recordings were made from the same neuron of a healthy control mouse before and after incubation with Hm1a (10 nM). Horizontal scale bar, 250 ms; vertical scale bar, 20 mV. Middle and right, group data show no effect of Hm1a application on electrical excitability in a sub-population (6 out of 11) of colonic DRG neurons. c, Left, representative high-threshold mechanoreceptive colonic fibres from CVH mice showing no change after application of Hm1a (100 nM). Middle, group data from Hm1a-non-responsive colonic fibres (4 out of 11). Right, group data showing a subpopulation of CVH colonic afferents (3 out of 10) unaltered by ICA-121432 in the presence or absence of Hm1a. d, Left, representative Hm1a-non-responsive colonic DRG neuron in whole-cell current clamp. Middle and right, group data show electrical excitability is unaltered by Hm1a in a subset (4 out of 11) of CVH colonic DRG neurons. Error bars represent mean ± s.e.m. No significant differences were observed using Student’s t-test (ad, middle; b, d, right) or one-way ANOVA with post hoc Bonferroni test (a, c, right).

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This file contains Supplementary Table 1, a list of venoms that produced no detectible or specific calcium response in cultured sensory neurons. (PDF 184 kb)

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Osteen, J., Herzig, V., Gilchrist, J. et al. Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain. Nature 534, 494–499 (2016).

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