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: 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: Tonic block of mouse neuronal or human Cav3.X [T-type] calcium channels induced by either 1 μM application of ProTx I or 1 μM application of ProTx II. For recordings from native channels, ProTx I was tested on mouse thalamic neurons, ProTx II was tested on mouse DRG neurons, which respectively, expressed Cav3.1 and Cav3.2 channels. Shown are representative current traces from these experiments showing similar ProTx I block of native thalamic mouse T-type currents (first trace) and recombinant human Cav3.1 (second trace), and similar ProTx II block of mouse DRG T-type current (third trace) and recombinant human Cav3.2 (fourth trace) (control traces are depicted in black). Error bars reflect standard errors, asterisks denote statistical significance relative to either hCav3.1 [ProTx I] or hCav3.2 [ProTx II] [*p < 0.05, **p < 0.01]. Currents were elicited by stepping from a holding potential of −110 mV to a test potential of −20 mV.

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, Recombinant, Control, Patch Clamp

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