Journal: Nature Communications

Article Title: Spider venom-derived peptide induces hyperalgesia in Na v 1.7 knockout mice by activating Na v 1.9 channels

doi: 10.1038/s41467-020-16210-y

Figure Lengend Snippet: a – c Current-clamp recordings show that HpTx1 decreases the membrane excitability of small DRG neurons from Na v 1.9-KO mice. a Bars show no significant changes in RMP (left, n = 29) or AP amplitude (right, n = 25), but a significant increase in rheobase (middle, n = 25, nonparametric Wilcoxon matched-pair signed-rank two-tailed test: P = 0.008) in the presence of 0.75 μM HpTx1. b AP traces recorded from a representative small Na v 1.9-KO DRG neuron before (black) and after (red) application of 0.75 μM HpTx1. The dashed lines indicate 0 mV. c Statistics plots show significant decreases in AP spike number in the presence of 0.75 μM HpTx1 ( n = 25, two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test, treatment × inject current: F (7,168) = 8.834, P < 0.0001; treatment: F (1,24) = 25.49, #### P < 0.0001; inject current: F (7,168) = 25.28, P < 0.0001). d Comparison of nocifensive behaviors (licking or biting) following intraplantar injection of vehicle (10 μl 0.9% saline, n = 6) versus HpTx1 (1 μM or 10 μM in 10 μl saline, n = 6) (two-way ANOVA followed by Tukey’s multiple comparisons test, treatment × genotype: F (2,30) = 8.551, P = 0.0012; treatment: F (2,30) = 11.04, P = 0.0003; genotype: F (1,30) = 24.37, P < 0.0001). e Mechanical response thresholds measured in paws in response to vehicle (black circles, n = 6), 1 μM HpTx1 (yellow squares, n = 6) or 10 μM HpTx1 (red triangles, n = 6) injections (two-way ANOVA followed by Tukey’s multiple comparisons test, treatment × genotype: F (2,30) = 18.68, P < 0.0001; treatment: F (2,30) = 0.0356, P = 0.9651; genotype: F (1,30) = 67.3, P < 0.0001). f Latency of WD to noxious heat stimuli measured after intraplantar injection of vehicle (black circles, n = 6), 1 μM HpTx1 (yellow squares, n = 6) or 10 μM HpTx1 (red triangles, n = 6) (two-way ANOVA followed by Tukey’s multiple comparisons test, treatment × genotype: F (2,30) = 44.54, P < 0.0001; treatment: F (2,30) = 9.701, P = 0.0006; genotype: F (1,30) = 113.5, P < 0.0001). All DRG neurons recorded were held at −53 ± 2 mV. Data are presented as the mean ± S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P ( c – f ) are presented in Supplementary Data . Source data are provided as a . .

Article Snippet: The sections were permeabilized in PBS containing 0.5% TritonX-100) for 10 min, and were blocked with 10% goat serum for 1 h. The sections were incubated for 24 h at 4 °C with polyclonal rabbit anti-Na v 1.9 (1:200; alomone labs).

Techniques: Two Tailed Test, Injection

Journal: Nature Communications

Article Title: Spider venom-derived peptide induces hyperalgesia in Na v 1.7 knockout mice by activating Na v 1.9 channels

doi: 10.1038/s41467-020-16210-y

Figure Lengend Snippet: a Sequence alignments corresponding to the DIV s3b-s4 region of Na v subtypes. The highlighted sequences show the regions swapped between Na v 1.8 and Na v 1.9. b Representative current traces from Na v 1.9/1.8 DIV s3b-s4 P1 (top) and Na v 1.8/1.9 DIV s3b-s4 P1 (bottom) chimaera channels in the absence (black) and presence (red) of HpTx1. c Effects of HpTx1 on WT and mutant hNa v 1.9 channels. Dot plots display the effect of 0.75 μM HpTx1 on the peak current (top, n = 14 for WT; n = 4 for T1444L, M1445L, I1446F, and T1448A; n = 5 for L1449I and E1450L; n = 3 for N1451K) and the persistent current (bottom, n = 14 for WT; n = 4 for T1444L, I1446F, T1448A, and N1451K; n = 5 for M1445L, L1449I, and E1450L). Key residues involved in the interaction between HpTx1 and hNa v 1.9 are labeled (one-way ANOVA with Dunnett’s multiple comparison test, I 95 /I peak : F (7,35) = 17.72, P < 0.0001; I/I max : F (7,38) = 8.157, P < 0.0001). d (top) Sequence alignments corresponding to the DII s3b-s4 region of Na v subtypes. The highlighted sequences show the regions swapped between Na v 1.7 and Na v 1.8. Representative current traces from Na v 1.7/1.8 DII s3b-s4 (bottom left) and Na v 1.8/1.7 DII s3b-s4 (bottom right) chimaera channels in the absence (black) or presence of 5 μM HpTx1 (red). e Dose-dependent inhibitory curves show the effect of HpTx1 on WT ( n = 7) and mutant hNa v 1.7 channels ( n = 4 for F813S, n = 6 for L814A and A815S, n = 3 for D816K, n = 6 for V817K, n = 7 for E818G, n = 4 for E818R, n = 5 for G819S and n = 3 for Na v 1.7/1.8 DII s3b-s4) and the Na v 1.8/1.7 DII s3b-s4 chimaera channel ( n = 5). f Bars show the fold changes in IC 50 values of HpTx1 for mutant channels compared with that for the WT hNa v 1.7 channel. Data are presented as the mean ± S.E.M. Exact P ( c ) are presented in Supplementary Data . Source data are provided as a . .

Article Snippet: The sections were permeabilized in PBS containing 0.5% TritonX-100) for 10 min, and were blocked with 10% goat serum for 1 h. The sections were incubated for 24 h at 4 °C with polyclonal rabbit anti-Na v 1.9 (1:200; alomone labs).

Techniques: Sequencing, Mutagenesis, Labeling