protxii  (Biosynth Carbosynth)

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