Journal: Molecular Brain

Article Title: Block of T-type calcium channels by protoxins I and II

doi: 10.1186/1756-6606-7-36

Figure Lengend Snippet: Summary of biophysical parameters of various T-type calcium channels in the absence or presence of A, 1 μ M ProTx II and B, 1 μ M ProTx I

Article Snippet: Both ProTx I and ProTx II were purchased from Alomone Labs (Jerusalem, Israel) and were dissolved in external recording solution at the stock concentration of 1 mM.

Techniques: Blocking Assay

Journal: The Journal of Headache and Pain

Article Title: Botulinum toxin type A attenuates trigeminal neuralgia-like pain by suppressing CGRP release and modulating NaV1.7-associated signaling

doi: 10.1186/s10194-026-02454-4

Figure Lengend Snippet: BoNT/A reduces voltage-gated sodium currents in TG neurons predominantly through NaV1.7-associated components rather than NaV1.8-associated components. ( a, b ) Representative whole-cell voltage-clamp traces of total voltage-gated sodium (Na V ) currents recorded from primary cultured TG neurons in the absence ( a ) or presence ( b ) of BoNT/A (100 nM). Currents were evoked by step depolarizations from − 80 mV to + 40 mV. ( c ) Quantification of the maximum peak Na V current amplitude between Control and BoNT/A-treated neurons. ( d ) Current–voltage (I–V) relationships of peak Na V currents. ( e ) Normalized conductance–voltage (G/G_max) activation curves of Na V channels. ( f ) Representative Na v current traces from TG neurons treated with ProTXII (NaV1.7-selective inhibitor, 100 nM) and VX-548 (NaV1.8-selective inhibitor, 1 µM). ( g ) Quantification of peak Na V current amplitude, showing robust suppression by ProTXII and VX-548. ( h ) I–V relationships of peak Na V currents, demonstrating a depolarizing shift and reduced current density following ProTXII/VX-548 treatment. ( i ) G/G_max activation curves, revealing a slight depolarizing shift in the voltage dependence of Na V channel activation. ( j ) Pie chart illustrating the relative contributions of NaV1.7 (ProTXII-sensitive) and NaV1.8 (VX-548-sensitive) components to the total Na V current inhibition. ( k ) Representative Na V current traces from TG neurons pre-treated with BoNT/A followed by co-administration of ProTXII and VX-548. ( l ) Quantification of peak Na V current amplitude, showing that BoNT/A pre-treatment significantly augmented the inhibitory effect of ProTXII/VX-548. ( m ) I–V relationships of peak Na V currents under combined treatment. ( n ) G/G_max activation curves, showing a more pronounced hyperpolarizing shift indicative of further suppression of channel open probability. ( o ) Pie chart illustrating the relative contributions to the inhibitory effect under combined treatment: NaV1.7-associated, NaV1.8-associated, and other mechanisms. Data are presented as mean ± SEM. n = 5 neurons per group; one-way ANOVA followed by Tukey’s post hoc test

Article Snippet: The cells were cultured in the medium containing 1 μM OL (Sigma, ST9H9BC1D2C1, USA) or 10 μM PD98059 (Sigma, P215, USA) for 1 h, and then 100 nM ProTxII (MCE, HY-P1221, USA) or 1 μM VX-548 (MCE, HY-148800, USA) was added for another 1 h of culture.

Techniques: Cell Culture, Control, Activation Assay, Inhibition

Journal: The Journal of Headache and Pain

Article Title: Botulinum toxin type A attenuates trigeminal neuralgia-like pain by suppressing CGRP release and modulating NaV1.7-associated signaling

doi: 10.1186/s10194-026-02454-4

Figure Lengend Snippet: NaV1.7 expression and function are regulated by CGRP signaling in TG neurons. ( a ) Representative fluorescence images of intracellular Ca 2+ (red) in primary cultured TG neurons from Control and olcegepant (OL, 1 µM)-treated groups, captured under baseline (0 s) and capsaicin (CAP, 1 µM) stimulation conditions. ( b ) Quantification of Ca 2+ fluorescence intensity changes (ΔF/F 0 ) in TG neurons. n = 50 neurons per group. ( c ) Representative immunofluorescence images of NaV1.7 (green) co-stained with DAPI (blue, nuclear counterstain) in TG neurons from Control and OL-treated groups. ( d ) Quantification of NaV1.7 relative fluorescence intensity. ( e ) Relative mRNA expression of Scn9a (encoding NaV1.7) in TG tissue, normalized to GAPDH. ( f ) Representative Western blot images of NaV1.7 in TG tissue, with GAPDH as the loading control. ( g ) Densitometric quantification of NaV1.7 protein levels, normalized to GAPDH. ( h ) Representative whole-cell voltage-clamp traces of Na V currents recorded from TG neurons in Control, OL, and OL + ProTXII (100 nM) groups. Currents were evoked by step depolarizations from − 80 mV to + 40 mV. ( i ) Current–voltage (I–V) relationships of peak Na V currents. ( j ) Normalized conductance–voltage (G/G_max) activation curves of Na v channels. ( k ) Quantification of the maximum peak Na V current amplitude. Data are presented as mean ± SEM. n = 3–5 neurons per group; one-way ANOVA followed by Tukey’s post hoc test

Article Snippet: The cells were cultured in the medium containing 1 μM OL (Sigma, ST9H9BC1D2C1, USA) or 10 μM PD98059 (Sigma, P215, USA) for 1 h, and then 100 nM ProTxII (MCE, HY-P1221, USA) or 1 μM VX-548 (MCE, HY-148800, USA) was added for another 1 h of culture.

Techniques: Expressing, Fluorescence, Cell Culture, Control, Immunofluorescence, Staining, Western Blot, Activation Assay

Journal: The Journal of Headache and Pain

Article Title: Botulinum toxin type A attenuates trigeminal neuralgia-like pain by suppressing CGRP release and modulating NaV1.7-associated signaling

doi: 10.1186/s10194-026-02454-4

Figure Lengend Snippet: BoNT/A suppresses ERK phosphorylation to reduce NaV1.7 membrane trafficking and is associated with lysosome-dependent downregulation of NaV1.7 in TG neurons. ( a–c ) Relative mRNA expression of Mapk14 (encoding p38, A ), Mapk3 (encoding ERK1, b ), and Mapk1 (encoding ERK2, c ) in TG tissue, quantified by qPCR and normalized to β-actin. ( d ) Representative Western blot images of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 in TG tissue from the three groups, with β-actin as the loading control. ( e ) Densitometric quantification of the p-ERK1/2 / total ERK1/2 ratio. ( f ) Representative Western blot images of p-ERK1/2 and total ERK1/2 in primary cultured TG neurons before and after α-CGRP application, with GAPDH as the loading control. ( g ) Densitometric quantification of the p-ERK1/2 / total ERK1/2 ratio corresponding to ( f ). ( h ) Representative immunofluorescence images of NaV1.7 (green) in TG sections from Control and PD98059 (ERK1/2 inhibitor, 10 µM)-treated groups, with DAPI (blue) as the nuclear counterstain. ( i ) Quantification of NaV1.7 relative fluorescence intensity. ( j ) Representative immunofluorescence images showing colocalization of LAMP1 (red, lysosomal marker) and NaV1.7 (green) in TG neurons from Control, BoNT/A-treated, and BoNT/A + Bafilomycin A1-treated groups. DAPI (blue) labels nuclei. ( k–m ) Fluorescence intensity profiles along the white dashed lines in ( j ), illustrating the colocalization correlation between LAMP1 and NaV1.7 in Control ( K ), BoNT/A ( l ), and BoNT/A + Bafilomycin A1 ( m ) groups. ( n ) Representative whole-cell voltage-clamp traces of Na V currents recorded from TG neurons treated with vehicle (Control), PD98059 (10 µM), or PD98059 combined with ProTXII (100 nM). Currents were evoked by step depolarizations from − 80 mV to + 40 mV. ( o ) Current–voltage (I–V) relationships of peak Na V currents. ( p ) Quantification of the maximum peak Na V current amplitude. Data are presented as mean ± SEM. n = 3–10 neurons per group; one-way ANOVA followed by Tukey’s post hoc test

Article Snippet: The cells were cultured in the medium containing 1 μM OL (Sigma, ST9H9BC1D2C1, USA) or 10 μM PD98059 (Sigma, P215, USA) for 1 h, and then 100 nM ProTxII (MCE, HY-P1221, USA) or 1 μM VX-548 (MCE, HY-148800, USA) was added for another 1 h of culture.

Techniques: Phospho-proteomics, Membrane, Expressing, Western Blot, Control, Cell Culture, Immunofluorescence, Fluorescence, Marker

Journal: Pain

Article Title: Analgesic treatment of ciguatoxin-induced cold allodynia.

doi: 10.1016/j.pain.2013.06.015

Figure Lengend Snippet: Fig. 2. Characterisation of ciguatoxin-induced responses in the human neuroblastoma cell line SH-SY5Y. (A) In SH-SY5Y cells loaded with Calcium-4 dye, stimulation with P- CTX-1 (EC50 2.2 ± 0.6 nM), P-CTX-2 (EC50 9.3 ± 2.6 nM), P-CTX-3 (EC50 8.3 ± 2.4 nM), and BTX-A (EC50 160.7 ± 19.3 nM) caused concentration-dependent increases in intracellular Ca2+. (B) P-CTX-1 responses were mediated through TTX-sensitive Nav isoforms endogenously expressed in SH-SY5Y cells, as responses were completely abolished in the presence of TTX (300 nM). (C) TTX completely inhibited P-CTX-1 responses with an IC50 of 12.9 ± 2.2 nM, while the Nav1.2 inhibitor TIIIA caused partial (25.4 ± 0.9%) inhibition with an IC50 of 49.9 ± 14.9 nM. The Nav1.7 inhibitor ProTxII caused a small inhibition (11.9 ± 1.6%) at concentrations (100 nM) that fully inhibit Nav1.7, and blocked P-CTX-1 responses with an IC50 of 4.3 ± 3.1 lM. (D) The Cav inhibitors nifedipine and CVID partially blocked P-CTX-1 responses with IC50s of 59.1 ± 16.4 nM and 33.6 ± 8.8 nM, respectively. Data are presented as mean ± SEM and is representative of 3 to 9 independent experiments.

Article Snippet: Veratridine was obtained from Ascent Scientific (Bristol, UK), tetrodotoxin (TTX) was from Enzo Life Sciences (Farmingdale, NY), and ProTxII was from Peptides International (Louisville, KY).

Techniques: Concentration Assay, Inhibition

Journal: Pain

Article Title: Analgesic treatment of ciguatoxin-induced cold allodynia.

doi: 10.1016/j.pain.2013.06.015

Figure Lengend Snippet: Fig. 5. Nav isoforms contributing to ciguatoxin-induced cold allodynia. Ciguatoxin- induced cold allodynia was not significantly inhibited in Nav1.3/ animals, or after intraplantar administration of the Nav1.7-specific inhibitor ProTxII (10 nM) and the Nav1.2/Nav1.1 inhibitor TIIIA (10 lM). Coadministration of TIIIA (10 lM) and the Nav1.8-inhibitor A803467 (10 lM) partially decreased cold allodynia elicited by intraplantar administration of P-CTX-1. Intraplantar administration of the Nav1.1/ Nav1.6 inhibitor GIIIA (10 lM) inhibited cold allodynia by 46.6 ± 8.9% and was additive to inhibition of Nav1.8, with coadministration of GIIIA and A803467 reducing cold pain behaviour by 87.2 ± 3.3%. Data are presented as mean ± SEM of 5 to 12 animals. Statistical significance was determined by ANOVA with Dunnett’s posttest. ⁄P < .05, ⁄⁄⁄P < .001.

Article Snippet: Veratridine was obtained from Ascent Scientific (Bristol, UK), tetrodotoxin (TTX) was from Enzo Life Sciences (Farmingdale, NY), and ProTxII was from Peptides International (Louisville, KY).

Techniques: Inhibition

Journal: Scientific Reports

Article Title: Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor

doi: 10.1038/srep39662

Figure Lengend Snippet: Potency and selectivity of synthetic ProTX-II and JNJ63955918.

Article Snippet: Synthetic ProTX-II (PTX-4450-s) and ziconotide were purchased from Peptides international (Louisville, KY, USA).

Techniques:

Journal: Molecular Pain

Article Title: Characterisation of Nav1.7 functional expression in rat dorsal root ganglia neurons by using an electrical field stimulation assay

doi: 10.1177/1744806917745179

Figure Lengend Snippet: ProTx-II blocks sodium channel currents in DRG neurons. (a) Whole cell patch-clamp recordings in DRG and hippocampal neurons were performed to investigate the effect of TTX and ProTx-II on sodium currents (elicited by a test voltage step of 25 ms duration from −80 mV holding potential to −20 mV). Representative sodium current traces from DRG and hippocampal neurons are shown before (black line) and after application of 100 nM TTX (top panel, blue trace) and 300 nM ProTx-II (bottom panel, red trace) for a total duration of 5 min. (b) Averaged data for the experiment in (a) showing normalised amplitude of sodium currents in DRG and hippocampal neurons after incubation with ProTx-II (300 nM) and TTX (100 nM); n = 3–6 cells, ***p < 0.001.

Article Snippet: TTX was purchased from Tocris (Bristol, UK) and ProTx-II from PeptaNova (Sandhausen, Germany).

Techniques: Patch Clamp, Incubation