synthetic pctx1  (Alomone Labs)


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    Structured Review

    Alomone Labs synthetic pctx1
    ( 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. <t>PcTx1</t> (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 .
    Synthetic Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synthetic pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synthetic pctx1 - by Bioz Stars, 2023-03
    86/100 stars

    Images

    1) Product Images from "Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition"

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

    Journal: eLife

    doi: 10.7554/eLife.73384

    ( 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 .
    Figure Legend 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 .

    Techniques Used: Labeling, Activation Assay, Concentration Assay, Fluorescence

    ( 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).
    Figure Legend 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).

    Techniques Used: Fluorescence

    ( 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 .
    Figure Legend 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 .

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

    ( 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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Fluorescence, Activation Assay

    ( 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 .
    Figure Legend 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 .

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

    ( 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.
    Figure Legend 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.

    Techniques Used: Activation Assay, Concentration Assay, Inhibition

    ( 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 .
    Figure Legend 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 .

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

    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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Mutagenesis

    synthetic pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
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    Structured Review

    Alomone Labs synthetic pctx1
    (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, highlighted in red. <t>PcTx1</t> (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 absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (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. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (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). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.
    Synthetic Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synthetic pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synthetic pctx1 - by Bioz Stars, 2023-03
    86/100 stars

    Images

    1) Product Images from "Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition"

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

    Journal: bioRxiv

    doi: 10.1101/2021.06.21.449215

    (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, 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 absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (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. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (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). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.
    Figure Legend Snippet: (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, 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 absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (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. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (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). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.

    Techniques Used: Activation Assay, Concentration Assay, Fluorescence

    (A) VCF trace of K105C* showing that the channel readily returns to a functional ‘apo’ state (current, black trace) after application of 300 nM PcTx1 at pH 7.4, while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace). (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). (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 in the presence of PcTx1 and leads to conformational rearrangements in both ASIC1a ECD and 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 PxTx1 is absent in the extracellular solution. Teal background shading indicates the presence of PcTx1 in the extracellular solution. Transitions between the binding modes that are explicitly shown in this work are indicated in full opacity. (E) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s BigDyn (1 μM) application (purple bar), with subsequent washout and activation. BigDyn is then applied again after the ‘ECD only ’ state is evoked through PcTx1 (300 nM) application, this time only resulting in a slow 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; and of the fluorescence signal induced by PcTx1 at pH 7.4 (Control) and BigDyn pre-application (Post BigDyn), respectively. Scale bars are 60 s (black horizontal), 10 μA (black vertical), and 10% (A and E) or 5 % (B) (red, A, B and E only). Data in C and F are presented as mean + 95CI.
    Figure Legend Snippet: (A) VCF trace of K105C* showing that the channel readily returns to a functional ‘apo’ state (current, black trace) after application of 300 nM PcTx1 at pH 7.4, while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace). (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). (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 in the presence of PcTx1 and leads to conformational rearrangements in both ASIC1a ECD and 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 PxTx1 is absent in the extracellular solution. Teal background shading indicates the presence of PcTx1 in the extracellular solution. Transitions between the binding modes that are explicitly shown in this work are indicated in full opacity. (E) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s BigDyn (1 μM) application (purple bar), with subsequent washout and activation. BigDyn is then applied again after the ‘ECD only ’ state is evoked through PcTx1 (300 nM) application, this time only resulting in a slow 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; and of the fluorescence signal induced by PcTx1 at pH 7.4 (Control) and BigDyn pre-application (Post BigDyn), respectively. Scale bars are 60 s (black horizontal), 10 μA (black vertical), and 10% (A and E) or 5 % (B) (red, A, B and E only). Data in C and F are presented as mean + 95CI.

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

    (A) Model of the co-crystal structure of cASIC1a and PcTx1 (teal) binding to the extracellular domain (PDBID 4FZO). Inset shows a close up of the interaction site at the acidic pocket, including ASIC1a residue F350 (orange). (B) Representative TEVC trace showing mASIC1a F350L pH activation in the absence (top) and presence (bottom) of 30 nM PcTx1. (C) Activation and SSD curve of F350L mASIC1a without (orange) and with (teal) PcTx1 (n=5–12). (D) TEVC traces showing the effect of 1 nM PcTx1 on mASIC1a WT (top) and 100 nM PcTx1 on F350L (bottom) applied at pH 7.4. (E) PcTx1 concentration-response curve at pH 7.4 using the protocol shown in (D) (n=4–14). (F) Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.4. (G) Left: Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.3. Right: Comparison of the fluorescence change upon PcTx1 application and after a 3 min washout between K105C* and K150C*F350L (H) Left: Representative trace of a VCF recording of V80C*F350L equivalent to the one shown in G. Right: Same analysis as in G but compared between V80C* and V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B and D only) and 10 μA (black vertical), and 10% (red) (F-H). Data in C, E, G and H are presented as mean + 95CI.
    Figure Legend Snippet: (A) Model of the co-crystal structure of cASIC1a and PcTx1 (teal) binding to the extracellular domain (PDBID 4FZO). Inset shows a close up of the interaction site at the acidic pocket, including ASIC1a residue F350 (orange). (B) Representative TEVC trace showing mASIC1a F350L pH activation in the absence (top) and presence (bottom) of 30 nM PcTx1. (C) Activation and SSD curve of F350L mASIC1a without (orange) and with (teal) PcTx1 (n=5–12). (D) TEVC traces showing the effect of 1 nM PcTx1 on mASIC1a WT (top) and 100 nM PcTx1 on F350L (bottom) applied at pH 7.4. (E) PcTx1 concentration-response curve at pH 7.4 using the protocol shown in (D) (n=4–14). (F) Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.4. (G) Left: Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.3. Right: Comparison of the fluorescence change upon PcTx1 application and after a 3 min washout between K105C* and K150C*F350L (H) Left: Representative trace of a VCF recording of V80C*F350L equivalent to the one shown in G. Right: Same analysis as in G but compared between V80C* and V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B and D only) and 10 μA (black vertical), and 10% (red) (F-H). Data in C, E, G and H are presented as mean + 95CI.

    Techniques Used: Binding Assay, Activation Assay, Concentration Assay, Mutagenesis, Fluorescence

    (A) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two or all three subunits. (B) Representative 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 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). Data points in E-G represent mean and 95CI. All scale bars are 4 μA and 60 s (B and D) or 30 s (F), respectively.
    Figure Legend Snippet: (A) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two or all three subunits. (B) Representative 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 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). Data points in E-G represent mean and 95CI. All scale bars are 4 μA and 60 s (B and D) or 30 s (F), respectively.

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

    (A) Representative VCF trace of 300 nM PcTx1 application to a concatemeric construct labelled 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 carrying 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 grey). (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 grey). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red). In D and E, errors are 95CI, unpaired t-Mann-Whitney test to neighbouring bar on the left, *P<0.05, **P<0.005, ***P<0.0005.
    Figure Legend Snippet: (A) Representative VCF trace of 300 nM PcTx1 application to a concatemeric construct labelled 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 carrying 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 grey). (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 grey). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red). In D and E, errors are 95CI, unpaired t-Mann-Whitney test to neighbouring bar on the left, *P<0.05, **P<0.005, ***P<0.0005.

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

    Schematic representation of a sideview of ASIC1a ECD and 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 colouring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ conformation 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.
    Figure Legend Snippet: Schematic representation of a sideview of ASIC1a ECD and 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 colouring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ conformation 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.

    Techniques Used: Binding Assay, Mutagenesis

    synthetic pctx1  (Alomone Labs)


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    Structured Review

    Alomone Labs synthetic pctx1
    Mechanism of ASIC1a modulation by BigDyn. (A) Amino acid sequences of BigDyn, DynA and DynB. (B/C) Representative current traces (B) and averaged data (C) obtained by pH 5.6 application (black bar in (B)) at mASIC1a WT-expressing Xenopus laevis oocytes after preincubation in pH 7.1 (grey bar) with or without 1 µM of the indicated peptide or peptide combination (green bar); in (B) control currents (after pH 7.4 conditioning) are shown in grey for comparison. Asterisk in (C) indicates significant difference to control condition (p < 0.0001); n = 5-68). (D) Concentration-response curves for activation (Act.) and steady-state desensitization (SSD) of WT ASIC1a in the presence and absence of 0.1 µM BigDyn (n = 4-15). (E) Structure of cASIC1a (PDB: 4NTW) with individual subunits color coded and inset showing the location of Lys105. (F) Representative current (black) and fluorescence (red) traces obtained by application of pH 5.5 (black bars) at mASIC1a labeled with Alexa Fluor 488 at position 105, the indicated peptide <t>(PcTx1,</t> 0.3 µM: blue bar; BigDyn, 1 µM: green bar; DynA, 10 µM: dark green bar, and DynB, 10 µM: light green bar) or pH 9.0 (purple bar). (G) Averaged change in fluorescence obtained by application of PcTx1, BigDyn, DynB or pH 9.0, as shown in (F) (normalized to that obtained by application of pH 5.5) (n = 5-15). Error bars in (C), (D) and (G) represent 95CI.
    Synthetic Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mechanism and site of action of big dynorphin on ASIC1a"

    Article Title: Mechanism and site of action of big dynorphin on ASIC1a

    Journal: bioRxiv

    doi: 10.1101/816264

    Mechanism of ASIC1a modulation by BigDyn. (A) Amino acid sequences of BigDyn, DynA and DynB. (B/C) Representative current traces (B) and averaged data (C) obtained by pH 5.6 application (black bar in (B)) at mASIC1a WT-expressing Xenopus laevis oocytes after preincubation in pH 7.1 (grey bar) with or without 1 µM of the indicated peptide or peptide combination (green bar); in (B) control currents (after pH 7.4 conditioning) are shown in grey for comparison. Asterisk in (C) indicates significant difference to control condition (p < 0.0001); n = 5-68). (D) Concentration-response curves for activation (Act.) and steady-state desensitization (SSD) of WT ASIC1a in the presence and absence of 0.1 µM BigDyn (n = 4-15). (E) Structure of cASIC1a (PDB: 4NTW) with individual subunits color coded and inset showing the location of Lys105. (F) Representative current (black) and fluorescence (red) traces obtained by application of pH 5.5 (black bars) at mASIC1a labeled with Alexa Fluor 488 at position 105, the indicated peptide (PcTx1, 0.3 µM: blue bar; BigDyn, 1 µM: green bar; DynA, 10 µM: dark green bar, and DynB, 10 µM: light green bar) or pH 9.0 (purple bar). (G) Averaged change in fluorescence obtained by application of PcTx1, BigDyn, DynB or pH 9.0, as shown in (F) (normalized to that obtained by application of pH 5.5) (n = 5-15). Error bars in (C), (D) and (G) represent 95CI.
    Figure Legend Snippet: Mechanism of ASIC1a modulation by BigDyn. (A) Amino acid sequences of BigDyn, DynA and DynB. (B/C) Representative current traces (B) and averaged data (C) obtained by pH 5.6 application (black bar in (B)) at mASIC1a WT-expressing Xenopus laevis oocytes after preincubation in pH 7.1 (grey bar) with or without 1 µM of the indicated peptide or peptide combination (green bar); in (B) control currents (after pH 7.4 conditioning) are shown in grey for comparison. Asterisk in (C) indicates significant difference to control condition (p < 0.0001); n = 5-68). (D) Concentration-response curves for activation (Act.) and steady-state desensitization (SSD) of WT ASIC1a in the presence and absence of 0.1 µM BigDyn (n = 4-15). (E) Structure of cASIC1a (PDB: 4NTW) with individual subunits color coded and inset showing the location of Lys105. (F) Representative current (black) and fluorescence (red) traces obtained by application of pH 5.5 (black bars) at mASIC1a labeled with Alexa Fluor 488 at position 105, the indicated peptide (PcTx1, 0.3 µM: blue bar; BigDyn, 1 µM: green bar; DynA, 10 µM: dark green bar, and DynB, 10 µM: light green bar) or pH 9.0 (purple bar). (G) Averaged change in fluorescence obtained by application of PcTx1, BigDyn, DynB or pH 9.0, as shown in (F) (normalized to that obtained by application of pH 5.5) (n = 5-15). Error bars in (C), (D) and (G) represent 95CI.

    Techniques Used: Expressing, Concentration Assay, Activation Assay, Fluorescence, Labeling


    Figure Legend Snippet:

    Techniques Used: Fluorescence

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    Alomone Labs synthetic pctx1
    ( 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. <t>PcTx1</t> (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 .
    Synthetic Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( 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 .

    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

    ( 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).

    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

    ( 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 .

    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

    ( 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.

    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

    ( 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 .

    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

    ( 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.

    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

    ( 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 .

    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

    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.

    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