asic1a specific inhibitor psalmotoxin 1 pctx1  (ApexBio)

Journal: Pflugers Archiv

Article Title: Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and K Ca 3.1 ion channels, but involves phosphoinositide 3-kinase

doi: 10.1007/s00424-022-02781-w

Figure Lengend Snippet: Enhanced migration of GSCs at acidic pH is not mediated by ASIC1a or ASIC3. a Inhibition of ASIC1 with 100 nM PcTx1 or of ASIC3 with 500 nM APETx2 did not affect migration at pH 7.4 or pH 6.6. n (from left to right) = 38, 28, 37, 29, 39, 33 individual spheres. b Sphere diameters between conditions were not significantly different from each other. c Activation of ASIC1a with 20 nM MitTx did not increase migration at pH 7.4 or pH 6.6. n (from left to right) = 14, 9, 13, 15. d Sphere diameters between conditions were not significantly different from each other. e Monoclonal R54 ASIC1a/b knockout cell lines migrated more aggressively at pH 6.6 than at pH 7.4. n (from left to right) = 26, 28, 22, 21, 29, 33. f Sphere diameters were not significantly different from each other. All experiments were performed in three biological replicates. Dots represent technical replicates. Bars represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Tukey’s test)

Article Snippet: The effect of ASICs on migration was tested using PcTx1 (Smartox Biotech, Saint-Egrevè, France), APETx2 (Alomone Labs, Jerusalem, Israel), or MitTx (Smartox Biotech) and the effect of KCNN4 and PI3K by TRAM-34 (Selleck Chemicals, Houston, USA) and wortmannin (Selleck Chemicals), respectively.

Techniques: Migration, Inhibition, Activation Assay, Knock-Out

Journal: Pflugers Archiv

Article Title: Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and K Ca 3.1 ion channels, but involves phosphoinositide 3-kinase

doi: 10.1007/s00424-022-02781-w

Figure Lengend Snippet: Overexpression of ASIC2a does not change aggressive migration of GSCs at acidic pH. a Pie chart indicating the relative occurrence of R54 cells with no ASIC current, ASIC currents elicited by pH 6 and by pH 5, and ASIC currents elicited only by pH 5. n = 9 for WT and n = 10 for ASIC2-over-expressing (oe) cells. b Left, representative current traces of wild-type and ASIC2 oe cells at pH 6 and pH 5. Right, ratio of ASIC currents elicited by pH 5 and pH 6. Error bars represent the mean ± SD; the mean value for ASIC2 oe cells was 9.23. n = 7–9. c Left, representative current traces of wild-type and ASIC2 oe cells at pH 6, in the absence and presence of PcTx1. Right, ratio of ASIC currents elicited by pH 6, in the presence of PcTx1. Error bars represent the mean ± SD; n = 7–8. d Sphere migration of R54 wild-type and ASIC2 oe cells. n (from left to right) = 99, 184, 157, 83. The experiment was performed in three biological replicates. Dots represent technical replicates. Bars represent mean ± SD. *** p < 0.001 (Tukey’s test). e Sphere diameters between conditions were not significantly different from each other. n = 30 for each condition

Article Snippet: The effect of ASICs on migration was tested using PcTx1 (Smartox Biotech, Saint-Egrevè, France), APETx2 (Alomone Labs, Jerusalem, Israel), or MitTx (Smartox Biotech) and the effect of KCNN4 and PI3K by TRAM-34 (Selleck Chemicals, Houston, USA) and wortmannin (Selleck Chemicals), respectively.

Techniques: Over Expression, Migration, Expressing

Journal: Physiological Research

Article Title: ASIC1a Involves the Acid-Mediated Activation of Pancreatic Stellate Cells Associated With Autophagy Induction

doi: 10.33549/physiolres.934950

Figure Lengend Snippet: Exposure to an acidic medium made the expression of ASIC1a in PSCs increase obviously. Immunofluorescence staining of ASIC1a (red) and cell nuclei (blue) (A). RT-PCR (B) and Western blotting result (C) of ASIC1a expression in control group (pH 7.4) and acidic medium group (pH 6.5). * P<0.05 (n=3).

Article Snippet: ASIC1a specific inhibitor Psalmotoxin-1 (PcTx1) was provided by ApexBio Technology.

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Physiological Research

Article Title: ASIC1a Involves the Acid-Mediated Activation of Pancreatic Stellate Cells Associated With Autophagy Induction

doi: 10.33549/physiolres.934950

Figure Lengend Snippet: ASIC1a involved the acid-induced activation of PSCs. The expression of α-SMA and collagen I in PSCs at different conditions was measured at mRNA level by RT-PCR (A–B) or at protein level by Western blotting (C). * P<0.05 (n=3). Repeated Western blotting experiments showed similar results. I (Control), II (pH 6.5), III (pH 6.5 + PcTx1), IV (pH 6.5 + ASIC1a siRNA), V (pH 6.5 + NC-siRNA).

Article Snippet: ASIC1a specific inhibitor Psalmotoxin-1 (PcTx1) was provided by ApexBio Technology.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Physiological Research

Article Title: ASIC1a Involves the Acid-Mediated Activation of Pancreatic Stellate Cells Associated With Autophagy Induction

doi: 10.33549/physiolres.934950

Figure Lengend Snippet: ASIC1a mediated the acid-induced autophagy of PSCs. After the corresponding treatment, autophagy-associated important molecules, Beclin 1, LC3B and p62 expression in PSCs were detected by RT-PCR (A–C) and Western blotting (D). * P<0.05 (n=3). Repeated Western blotting experiments showed similar results. I (Control), II (pH 6.5), III (pH 6.5 + PcTx1), IV (pH 6.5 + ASIC1a siRNA), V (pH 6.5 + NC-siRNA).

Article Snippet: ASIC1a specific inhibitor Psalmotoxin-1 (PcTx1) was provided by ApexBio Technology.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Physiological Research

Article Title: ASIC1a Involves the Acid-Mediated Activation of Pancreatic Stellate Cells Associated With Autophagy Induction

doi: 10.33549/physiolres.934950

Figure Lengend Snippet: Involvement of autophagy in ASIC1a mediated acid-induced PSCs activation. After treatment with autophagy inhibitor alone or together with ASIC1a knockdown, the expression of α-SMA and collagen I in PSCs was detected by RT-PCR (A–B) and Western blotting (C). * P<0.05 (n=3). Repeated Western blotting experiments showed similar results.

Article Snippet: ASIC1a specific inhibitor Psalmotoxin-1 (PcTx1) was provided by ApexBio Technology.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Cannabinoids Inhibit Acid-Sensing Ion Channel Currents in Rat Dorsal Root Ganglion Neurons

doi: 10.1371/journal.pone.0045531

Figure Lengend Snippet: Three types of proton-gated currents were recorded in DRG neurons. Example traces of a slow-inactivating current followed by a small sustained component (A), and a fast-inactivating current (B), a sustained current with a small transient phase (C). All three types of proton-induced currents could be partly inhibited by 10 nM PcTx1, a specific antagonist of homomeric ASIC1a channels. These currents could be completely or partly blocked by 100 µM amiloride (Amil), a broad-spectrum ASIC channel blocker. The TRPV1 blocker AMG 9810 (1 µM) has little effect on the transient peak phases of three types of acid currents. The bar graph in (D) shows relative amplitude of peak currents induced by pH 5.5 after application of amiloride, PcTx1 and AMG 9810. Proton-induced currents were evoked by extracellular application of a pH 5.5 solution for 5 s. *P<0.05,**P<0.01, ***P<0.001, one way analysis of variance followed by post hoc Bonferroni’s test, compared with control. Each column represents the mean ± SEM of 8–11 neurons.

Article Snippet: Drugs used in the experiments were purchased from Sigma Chemical Co. and include hydrochloric acid, WIN55,212-2, WIN55,212-3,AM281, forskolin, 8-Br-cAMP, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b] , dioxin-6-yl) acrylamide (AMG 9810), psalmotoxin 1 (PcTX1) and amiloride.

Techniques:

Journal: PLoS ONE

Article Title: Cannabinoids Inhibit Acid-Sensing Ion Channel Currents in Rat Dorsal Root Ganglion Neurons

doi: 10.1371/journal.pone.0045531

Figure Lengend Snippet: Intraplantar injection acetic acid (0.6%, 20 µl) evoked a flinch/shaking response. The bar graph in (A) shows acid-evoked pain was blocked by pretreatment of 200 µM amiloride, and partly blocked by pretreatment of 30 nM PcTx1. In contrast, the acid-evoked pain did not obviously change with pretreatment of 10 µM AMG 9810, *P<0.05, **P<0.01, unpaired t-test, compared with control. n = 8/each column. The bar graph in (B) shows the pretreatment of WIN55,212-2 (WIN) decreased flinching behavior induced by acetic acid in a dose-dependent manner. The effect of WIN55,212-2 was blocked by AM281 (10 −6 M), a selective CB1 receptor antagonist. *P<0.05, **P<0.01, one way analysis of variance followed by post hoc Bonferroni’s test, compared with control; & P<0.01, post hoc Bonferroni’s test, compared with WIN (10 −7 M) column. n = 8/each column. Flinching shaking of paw was recorded as the number of flinches per observation period (5 min).

Article Snippet: Drugs used in the experiments were purchased from Sigma Chemical Co. and include hydrochloric acid, WIN55,212-2, WIN55,212-3,AM281, forskolin, 8-Br-cAMP, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b] , dioxin-6-yl) acrylamide (AMG 9810), psalmotoxin 1 (PcTX1) and amiloride.

Techniques: Injection

Journal: PLoS ONE

Article Title: Expression and Activity of Acid-Sensing Ion Channels in the Mouse Anterior Pituitary

doi: 10.1371/journal.pone.0115310

Figure Lengend Snippet: A . Representative ASIC currents elicited by pH 5.5 with various concentrations of amiloride. Holding potential was −70 mV. The cells were held at pH 7.4 before and in between applications of pH 5.5. Amiloride was applied during acid application. B . Summary data showing the dose-dependent block of ASIC currents by amiloride in anterior pituitary cells (n = 7). Currents were normalized to the control condition (without amiloride). The IC 50 of amiloride block was 6.3±1.0 µM (n = 7). C. Representative ASIC currents in WT and ASIC2 -/- cells elicited by pH 5.5 with 100 nM PcTx1. Cells were pre-treated with PcTx1 in pH 7.4 for 1 min and then were perfused with pH 5.5 containing 100 nM PcTx1. Holding potential was −70 mV. D. Summary data showing the block of ASIC currents by 100 nM PcTx1 in anterior pituitary cells from WT (n = 7) and ASIC2 -/- mice (n = 6). * P <0.05 compared to control, Student's t -test.

Article Snippet: Psalmotoxin-1 (PcTX1) was ordered from Peptides International.

Techniques: Blocking Assay

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Labeling, Activation Assay, Concentration Assay, Fluorescence

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Representative trace of a VCF recording of K105C* showing application of PcTx1 (300 nM) washed off for 3 min with alternating pHs (20 s pH 7.4, 60 s pH 8.4, 40 s pH 7.4, 60 s pH 8.4) before switching to pH 7.4 again. ( B ) Comparison of the fluorescence of K105C* after a 3 min washout of 300 nM PcTx1 using pH 7.4, 8.4, or a mix of the two as shown in the protocol in ( A ) and . ( C ) Same as in ( A ) but for V80C* and running buffer 7.7 instead of 7.4. ( D ) Same as in ( B ) but for V80C* and base on the protocol shown in ( C ) and . All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( B ) and ( D ) are presented as mean ± 95 CI, ordinary analysis of variance (ANOVA).

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Fluorescence

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Voltage-clamp fluorometry (VCF) trace of K105C* showing the introduction of the ‘Global’ inhibitory binding mode upon application of 300 nM PcTx1 at pH 7.4. During washout and repeated activation, the channel readily returns to a functional apo state (current, black trace) while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace), characteristic for the ‘ECD only ’ state. ( B ) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle), and pH 8.0 (right). Respective PcTx1 binding modes are indicated below the traces. ( C ) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in ( B ) normalized to the fluorescence change induced by pH 5.5 application. ( D ) Schematic representation of the different pH-dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH that leads to conformational rearrangements in the extracellular domain (ECD) and the pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PcTx1 is absent in the extracellular solution. Teal background shading in the ‘Loose’ and ‘Global’ indicates the presence of PcTx1 in the extracellular solution (although not mandatory, see text for details). ( E ) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s big dynorphin (BigDyn) (1 µM) application (purple bar), with subsequent washout and activation. BigDyn is reapplied after the ‘ECD only ’ state has been evoked through PcTx1 (300 nM) application, this time resulting in a smaller decrease in the fluorescence signal. ( F ) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the apo (control) and to the PcTx1-induced ‘ECD only ’ state (post PcTx1), normalized to the signal induced by pH 5.5. ( G ) VCF trace of K105C* where 300 nM PcTx1 is applied to the ‘ECD only ’ state. ( H ) Quantitative analysis of the protocol shown in ( G ) comparing the fluorescence change induced by PcTx1 to the apo state at 7.4 (control) with the PcTx1 application to the ‘ECD only ’ state. All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( C ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 2—source data 1. VCF data from mASIC1a K105C* of single and multiple activations during PcTx1 washout, as shown in and . Figure 2—source data 2. VCF data from mASIC1a K105C* of PcTx1 application at pH 8.0 followed by different washout protocols, as seen in . Figure 2—source data 3. VCF data of mASIC1a K105C* of BigDyn and PcTx1 application, as seen in and .

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Binding Assay, Activation Assay, Functional Assay, Fluorescence

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Representative VCF trace of K105C* showing application of PcTx1 (300 nM) washout for 3 min before pH 5.5 stimulus. Corresponding binding modes are indicated below. ( B ) Comparison of the final pH 5.5-induced current (I) and fluorescence (ΔF) at pH 7.4 in recordings shown in , where channels undergo three 1 min washouts at pH 7.4, each followed by pH 5.5 stimulus (left) or the protocol shown in ( A ) with a single 3 min washout (right). The final pH 5.5-induced current was normalized to the one at the beginning of the recording, the fluorescence was analyzed right before the final pH 5.5 activation and normalized to the deflection induced by PcTx1. ( C ) Representative VCF trace of K105C* showing pH 7.0 stimuli at various states of the recording. Corresponding binding modes are indicated below. ( D ) Representative trace of a VCF recording of K105C* depicting 30 s pre-conditioning with big dynorphin (BigDyn) (1 μM) and subsequent 30 s PcTx1 (300 nM) application and pH 5.5 activation. ( E ) Quantitative comparison of the fluorescence change induced by 300 nM PcTx1 at pH 7.4 in the apo state (control) and after BigDyn (1 μΜ) application, normalized to the signal induced by pH 5.5. Data in ( B ) and ( E ) are presented as mean ± 95CI.

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Binding Assay, Fluorescence, Activation Assay

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two, or all three subunits. ( B ) Representative two-electrode voltage-clamp (TEVC) trace of activation ( C ) Activation curve from recordings shown in ( B ) for the four different concatemeric constructs (n = 7–13). ( D ) Representative TEVC trace of steady-state desensitization (SSD). ( E ) SSD profiles from recordings shown in ( D ) (n = 4–11). ( F ) Representative TEVC trace of concentration-dependent PcTx1 inhibition at pH 7.4. ( G ) PcTx1 concentration–response curves from data shown in ( F ) (n = 4–11). Scale bars are 4 µA (vertical) and 60 s (horizontal) for (B, D) and 30 s for (F). Data points in ( C, E and G ) represent mean ± 95CI. Figure 4—source data 1. TEVC data from concatemeric mASIC1a of pH-dependent activation, as shown in and . Figure 4—source data 2. TEVC data from concatemeric mASIC1a of SSD, as shown in . Figure 4—source data 3. TEVC data from concatemeric mASIC1a of PcTx1 concentration–response curve, as shown in and .

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Construct, Mutagenesis, Activation Assay, Concentration Assay, Inhibition

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Activation and steady-state desensitization (SSD) curves for trimeric and concatemeric WT and F350L channels in comparison. ( B ) Activation curves for all concatemeric variants showing that concatemers with the same number of F350L-bearing subunits cluster around similar pH sensitivities. ( C ) Same as in ( B ), but for concentration-dependent PcTx1 inhibition. Data are presented as mean ± 95 CI.

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Activation Assay, Concentration Assay, Inhibition

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Representative voltage-clamp fluorometry (VCF) trace of 300 nM PcTx1 application to a concatemeric construct labeled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. ( B ) Same as in ( A ) but one subunit carries a F350L mutation. ( C ) Same as in ( A ) but with two subunits carry a F350L mutation. ( D ) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in ( A – C ). Results from non-concatenated channels are indicated for comparison (shown in light gray). ( E ) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light gray). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red vertical). In ( D ) and ( E ), error bars represents 95CI, unpaired Mann–Whitney test to neighboring bar on the left, *p<0.05, **p<0.005, ***p<0.0005. Figure 5—source data 1. VCF data from concatemeric mASIC1a of PcTx1 application and washout as shown in .

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Construct, Labeling, Mutagenesis, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: Schematic representation of a side view of acid-sensing ion channel 1a (ASIC1a) extracellular domain (ECD) and transmembrane domain (TMD) and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view coloring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ state with increasing number of F350L-containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

Techniques: Binding Assay, Mutagenesis