Journal: bioRxiv

Article Title: A novel sodium signaling complex regulates uterine activity

doi: 10.1101/2020.07.31.229138

Figure Lengend Snippet: During the quiescent state, progesterone binding to the progesterone receptor (PR A/B ) increases NALCN expression and activity . Sodium current through NALCN activates SLO2.1 channels, increasing K + efflux to maintain the cell in a hyperpolarized state. As a result, voltage-dependent Ca 2+ channels (VDCCs) are closed, and uterine contractions do not occur. In the contractile state, estrogen acting on ERα inhibits NALCN expression , leading to decreased SLO2.1 activity. The reduced K + efflux depolarizes the membrane, leading to VDCC activation, an increase in intracellular Ca 2+ , and uterine contractility. At labor, Oxytocin (OXT) binds to the oxytocin receptor (OTR), leading to activation of phospholipase C (PLC), production of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), and production of inositol triphosphate (IP 3 ). IP 3 activates the release of Ca 2+ from intracellular stores, and PIP 2 activates protein kinase C (PKC), which inhibits SLO2.1 . This SLO2.1 inhibition further depolarizes the membrane, thus opening more VDCCs, increasing intracellular Ca 2+ , and further activating myosin to cause muscle contraction.

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq) and SLO2.1 (rabbit polyclonal, 1:200, Alomone).

Techniques: Binding Assay, Expressing, Activity Assay, Activation Assay, Inhibition

Journal: Scientific Reports

Article Title: Chronic antidepressant potentiates spontaneous activity of dorsal raphe serotonergic neurons by decreasing GABA B receptor-mediated inhibition of L-type calcium channels

doi: 10.1038/s41598-017-13599-3

Figure Lengend Snippet: L-type voltage-dependent Ca 2+ channel (VDCC) participates in the spontaneous activity of DRN serotonergic neurons. ( a ) Representative traces of the spontaneous firing before (left) and after (right) the application of BAY K 8644 (BAY K; 1 μM). ( b ) The effect of BAY K 8644 (BAY K; 1 μM) on the spontaneous firing rate in serotonergic neurons. The average firing rate between 150–180 s after beginning the perfusion was compared to the basal firing rate (right). * P < 0.05 vs . Control. (Control, n = 4 neurons from 2 mice; BAY K, n = 6 neurons from 3 mice; P = 0.0496, Student’s t -test). ( c ) To block L-type VDCC current only in the recorded neurons, D890 (0.5 mM) was added to pipette solution, and the spontaneous firing was recorded for 30 s both in cell-attached (left) and whole-cell (right) configurations. ( d ) The average firing rate between 60–90 s after beginning the whole-cell recordings was compared to that in the cell-attached recordings. * P < 0.05 vs . Control. (Control, n = 4 neurons from 3 mice; D890, n = 4 neurons from 3 mice; P = 0.0190, Student’s t -test). Data are presented as the mean ± S.E.M.

Article Snippet: 6,7-dinitroquinoxaline-2,3(1 H,4 H)-dione (DNQX; an AMPA (non-NMDA) antagonist; Tocris Bioscience, Bristol, UK), bicuculline (a selective GABA A antagonist; Enzo Life Science, Farmingdale, NY, USA), prazosin (an α 1 receptor antagonist; Sigma-Aldrich), ZD7288 (a selective hyperpolarization-activated cyclic nucleotide–gated channel blocker; Cayman Chemical Company, Ann Arbor, MI, USA), (S)-(-)-Bay K 8644 (Bay K 8644; an L-type voltage-dependent Ca 2+ channel (VDCC) activator; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nifedipine (an L-type VDCC blocker; Wako Pure Chemical Industries, Osaka, Japan), KT5720 (a selective PKA inhibitor; Wako Pure Chemical Industries), and gallein (a selective G βγ inhibitor; Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Activity Assay, Control, Blocking Assay, Transferring

Journal: Scientific Reports

Article Title: Chronic antidepressant potentiates spontaneous activity of dorsal raphe serotonergic neurons by decreasing GABA B receptor-mediated inhibition of L-type calcium channels

doi: 10.1038/s41598-017-13599-3

Figure Lengend Snippet: Chronic administration of citalopram increased L-type VDCC current. ( a ) Outline of recordings from citalopram-administrated mice. After chronic treatment with citalopram (Cit; 24 mg/kg/day) or its vehicle (Water) for 28 days, acute raphe slices were prepared, and whole-cell voltage clamp recordings were performed. ( b ) Representative traces (left) and peak current (right) of high voltage activated (HVA) current in DRN serotonergic neurons from drug-naïve (Water) and citalopram-treated (Cit) mice. * P < 0.05 vs . Water. (Water, n = 8 neurons from 3 mice; Cit, n = 9 neurons from 4 mice; Student’s t -test; P = 0.0487). ( c ) Effects of intracellularly applied D890 on HVA current in DRN serotonergic neurons. P = 0.3402 vs . Water by Student’s t -test, Water, n = 8 neurons from 2 mice; Cit, n = 8 neurons from 2 mice. ( d ) Effects of bath-applied CGP52432 on high voltage activated (HVA) current in DRN serotonergic neurons. P = 0.5283 vs . Water by Student’s t -test, Water, n = 14 neurons from 4 mice; Cit, n = 11 neurons from 2 mice. ( e ) HVA VDCC current was recorded in the presence of WAY100635 and GR127935. * P < 0.05 vs . Water. (Water, n = 11 neurons from 2 mice; Cit, n = 12 neurons from 2 mice; Student’s t -test; P = 0.0107). ( f ) Effects of intracellularly applied KT5720 on HVA current in DRN serotonergic neurons. P = 0.6866 vs . Water by Student’s t -test, Water, n = 10 neurons from 2 mice; Cit, n = 12 neurons from 2 mice. ( g ) Effects of intracellularly applied gallein (20 μM) on HVA current in DRN serotonergic neurons. * P < 0.05 vs . Water. (Water, n = 12 neurons from 2 mice; Cit, n = 17 neurons from 2 mice; Student’s t -test; P = 0.0172). Data are presented as the mean ± S.E.M. ( h ) In normal condition, continuous GABA B receptor signaling inhibits PKA activation. Decreased PKA activity might cause inhibition of L-type VDCC activity and subsequently serotonergic firing activity. After chronic antidepressant treatment, postsynaptic GABA B signaling is decreased, resulting in activation of PKA and disinhibition of L-type VDCCs. Increasing Ca 2+ current through L-type VDCCs accelerates serotonergic firing activity.

Article Snippet: 6,7-dinitroquinoxaline-2,3(1 H,4 H)-dione (DNQX; an AMPA (non-NMDA) antagonist; Tocris Bioscience, Bristol, UK), bicuculline (a selective GABA A antagonist; Enzo Life Science, Farmingdale, NY, USA), prazosin (an α 1 receptor antagonist; Sigma-Aldrich), ZD7288 (a selective hyperpolarization-activated cyclic nucleotide–gated channel blocker; Cayman Chemical Company, Ann Arbor, MI, USA), (S)-(-)-Bay K 8644 (Bay K 8644; an L-type voltage-dependent Ca 2+ channel (VDCC) activator; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nifedipine (an L-type VDCC blocker; Wako Pure Chemical Industries, Osaka, Japan), KT5720 (a selective PKA inhibitor; Wako Pure Chemical Industries), and gallein (a selective G βγ inhibitor; Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Activation Assay, Activity Assay, Inhibition

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: TaqMan™ gene expression assays used in this study

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Gene Expression

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: Functional TRPM3 expression in human dorsal root ganglion (DRG) neurons. (a) Expression levels of TRP channel mRNA relative to HPRT in DRG neurons from two donors, determined using RT‐qPCR. Access to fresh human DRG tissue is scarce, hence the limited number of samples in this exploratory experiment. nd, not detected. (b) Representative example of changes in Fluo‐8‐fluorescence in human DRG neurons in response to the TRPM3 agonists pregnenolone sulphate (PS; 50 μM) and CIM0216 (10 μM) and to the TRPV1 agonist capsaicin (Caps; 200 nM). A high K+ solution was applied at the end of the experiment to confirm neuronal excitability. A trace showing the mean ± SEM of the changes in Fluo‐8‐fluorescence for all cells in this particular experiment is provided in Figure S1A. (c) Pie chart showing the distribution of neurons responding to PS, capsaicin or both (n = 206 hDRG neurons). (d) Representative example showing the reversible inhibition of PS‐induced responses in hDRG neurons by the selective TRPM3 antagonist isosakuranetin (Isosa; 20 μM). A trace showing the mean ± SEM of the changes in Fluo‐8‐fluorescence for all cells in this particular experiment is provided in Figure S1B. (e) Normalized responses to repeated PS applications. Neurons were stimulated three times with PS, and the second application occurred in the presence of either isosakuranetin (10 μM; n = 50) or vehicle control (n = 39). *P < .05 (two‐way repeated measures ANOVA with Tukey's post hoc test) [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Functional Assay, Expressing, Quantitative RT-PCR, Fluorescence, Inhibition, Control

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: Properties of human stem cell‐derived sensory neurons (hSCDS) neurons. (a) Brightfield image of immunohistochemically stained hSCDS neurons with anti‐TRPM3 antibody and negative control (only secondary antibody) showing a neuronal morphology with cell body clusters interconnected by neurites. Brown peroxidase‐based TRPM3 staining is visible in cell bodies as well as neurites. Scale bar: 500 μm. (b) Whole‐cell current‐clamp recordings on hSCDS neurons demonstrate the ability to fire action potentials upon current injection. (c) RT‐qPCR comparing relative expression of sensory TRP channels, nociceptor marker genes, and pluripotency genes between human embryonic stem (hES) cells (black) and hSCDS neurons (red). Results represent mean ± SEM from five independent experiments with three technical replicates each. nd, not detected in hES cells human embryonic stem cells [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Derivative Assay, Staining, Negative Control, Injection, Quantitative RT-PCR, Expressing, Marker

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: Functional expression profile of somatosensory TRP channels in human stem cell‐derived sensory neurons (hSCDS) neurons. (a) Changes in intracellular calcium in single hSCDS neurons stimulated with the TRP channel agonists menthol (100 μM), MO (100 μM), capsaicin (Caps;1 μM), and pregnenolone sulphate (PS;40 μM) and with a high K+ solution to probe excitability. (b) Venn diagram showing the pattern of responses to TRP channel agonists in hSCDS neurons (n = 1,180 cells in five independent differentiations). (c) Intracellular calcium measurements showing reversible inhibition of PS‐evoked responses by isosakuranetin (5 μM). PS‐responsive hSCDS neurons also responded to the synthetic TRPM3 agonist CIM0216 (1 μM). (d) Quantification of calcium responses for experiments as in panel (c) (n = 72). Isosa, isosakuranetin; MO, mustard oil [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Functional Assay, Expressing, Derivative Assay, Inhibition

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: Electrophysiological properties of TRPM3 in human stem cell‐derived sensory neurons (hSCDS) neurons. (a) Time course of inward (at −120 mV) and outward (at +80 mV) whole‐cell currents in hSCDS neurons showing the effects of the TRPM3 agonists pregnenolone sulphate (PS; 40 μM) and CIM0216 (1 μM). (b) Quantification of the amplitude of inward and outward currents activated by PS and CIM0216 (n = 6). (c) IV relationships corresponding to the indicated time points in panel (a). (d) Difference currents illustrate the typical outward rectification of PS‐activated currents and double rectification of CIM0216‐activated currents. (e,f) CIM0216‐activated whole‐cell currents were partially and reversibly inhibited by isosakuranetin (ISOSA; 5 μM) [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: Pharmacological characterization of TRPM3 in human stem cell‐derived sensory neurons (hSCDS) neurons. (a) Time course of the intracellular Ca2+ concentration in PS responding hSCDS neurons upon increasing the concentration of pregnenolone sulphate (PS) from 1 to 300 μM. (b) Concentration dependence of the PS‐induced calcium response in hSCDS neurons. Responses were normalized to the maximal response (n = 117). Solid line represents a fit using a Hill function. (c) Normalized calcium traces showing the effect of increasing concentrations of the TRPM3 antagonist isosakuranetin (Isoas) on the response to PS (40 μM). Green trace represents the vehicle control, showing mild rundown of the signal in the absence of antagonist. (d) Concentration–response curve for the inhibition of PS‐evoked calcium responses by isosakuranetin (n = 93; red squares) and primidone (n = 61; black dots). The mean normalized calcium response of vehicle‐treated hSCDS neurons (green trace in panel (c)) was used as control. Solid line represents a fit using a Hill function [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Derivative Assay, Concentration Assay, Control, Inhibition

Journal: British Journal of Pharmacology

Article Title: Functional expression and pharmacological modulation of TRPM3 in human sensory neurons

doi: 10.1111/bph.14994

Figure Lengend Snippet: Modulation of TRPM3 in human stem cell‐derived sensory neurons (hSCDS) neurons by GPCRs. (a) Example intracellular calcium measurements showing variable degrees of inhibition of PS‐induced responses upon activation of endogenously expressed μ‐opioid receptors using DAMGO (300 nM). (b) Scatter plot showing the range of DAMGO‐induced inhibition of PS responses in hSCDS neurons. Red line represents the mean percentage of TRPM3 inhibition caused by DAMGO (data from six experiments, with hSCDS neurons from three different differentiations). (c) Example intracellular calcium traces showing inhibition of PS‐induced responses by the GABAB receptor agonist baclofen (25 μM). (d) Scatter plot showing the range of baclofen‐induced inhibition of PS. Red line represents the mean percentage of inhibition caused by baclofen (data from six experiments, with hSCDS neurons from two different differentiations) [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons . eLife , 6 10.7554/eLife.26138 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ] Rostock, C. , Schrenk‐Siemens, K. , Pohle, J. , & Siemens, J. (2018).

Techniques: Derivative Assay, Inhibition, Activation Assay

Journal: Science signaling

Article Title: B cell responses to membrane-presented antigens require the function of the mechanosensitive cation channel Piezo1

doi: 10.1126/scisignal.abq5096

Figure Lengend Snippet: (A) Purified human peripheral blood or tonsillar naive B cells were loaded with the Ca2+ indicator dye, CAL520, and placed in a glass chamber for 5 to 30 min, in the absence (Untreated) or presence of the Piezo1 inhibitor, GsMTx4. The cells were then touched with a glass probe and images were captured by confocal microscopy. Left: Images of the Ca2+ signals of two representative cells that were either untreated or treated with GsMTx4 before, during, and after contact with the glass probe. Middle: Quantification of the fold-increase in Ca2+ flux for each of the cells over the course of 40 s. Right: The average fold-change in Ca2+ flux (relative to that in cells at the time of first contact) in untreated B cells (36 cells) and GsMTx4-treated B cells (54 cells) over 13 s. Data are means ± SEM from three experiments. (B) Purified human tonsillar CD10− naïve and memory B cells were loaded with CAL520 and a red fluorescent dye as an indicator of cell volume and were placed on a planar lipid bilayer (PLB) or glass coverslips (Glass) and imaged over time by TIRFM. Top: Representative images of two B cells placed on PLB or glass and displayed as color-coded ratio images by normalizing CAL520 MFI with red fluorescence dye MFI and quantification of Ca2+ fluxes and contact areas with time after first contact with the PLB (26 cells) or glass (36 cells). Bottom: The fold-change in Ca2+ flux over time relative to the first point of contact of the cell with glass or PLB (left) and the area of contact of the indicated cells with the surface over time (right). Right: Comparison of the maximal Ca2+ flux increases of cells placed on PLB or glass, which were calculated for each cell by normalizing its maximal CAL520 MFI to the MFI upon first contact with PLB (33 cells) or glass (110 cells). Data are means ± SEM from one experiment that is representative of six experiments. (C) Tonsillar naïve B cells and memory B cells were left untreated or were pretreated with 20 μM OB-1 and analyzed by TIRFM on glass in the continued presence of OB-1. Left: Representative images of two cells (top) and quantification of changes in Ca2+ flux (bottom left) and cell area (bottom right) over time. Right: Comparison of the maximal Ca2+ flux increases and maximal area of cell spreading (Areamax) for cells placed on glass in the absence (27 cells) or presence (39 cells) of OB-1. Data are means ± SEM from one experiment that is representative of 11 experiments. (D) BCR− Ramos cells or Ramos cells transduced to stably express exogeneous IgM H and L chain (Trans BCR+) were loaded with CAL520 and red fluorescence dye and placed on glass for the indicated times while Ca2+ flux were recorded. Representative ratio images (left) and quantification of fold-change in Ca2+ flux for BCR− cells (31 cells) and Trans BCR+ cells (30 cells) over time. Data are means ± SEM from one experiment that is representative of three experiments. Scale bars, 5 μm. (B to D) Scale ranges of the 8-bit, color-coded image display. The kinetic data in (A) to (D) were tested for statistical significance (95% confidence interval) with the ‘compareGrowthCurves’ function (see Materials and Methods). **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ns, not significant. For (B and C, right), data were analyzed by two-tailed unpaired t-test with Welch’s correction. ***P ≤ 0.001 and ****P ≤ 0.0001.

Article Snippet: Reverse transcription and qRT-PCR qRT-PCR assays were performed with a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems), Cells-to-CT 1-Step Taqman Kit (Invitrogen, A25603), and GAPDH (NM_002046.3) and PIEZO1 (Hs00207230_m1) TaqMan Probes (Thermo Fisher).

Techniques: Purification, Confocal Microscopy, Fluorescence, Comparison, Stable Transfection, Two Tailed Test

Journal: Science signaling

Article Title: B cell responses to membrane-presented antigens require the function of the mechanosensitive cation channel Piezo1

doi: 10.1126/scisignal.abq5096

Figure Lengend Snippet: (A) Purified human tonsillar B cells were labeled with CAL520 and a monoclonal antibody specific for the GC B cell marker, CD10, to gate naïve and memory B cells. After 180 s of baseline recording (None, black), 15 μM Yoda1 (blue) was added to the B cells in solution, which was followed by the addition of 2 mM EGTA (red). Ca2+ signals were analyzed for 1200 s by flow cytometry. Left: Representative plot of the median CAL520 fluorescence intensity (FI) values over time. Right: Quantification of the Ca2+ influxes calculated for B cells from five donors as the fold-change in the median CAL520 FI induced by Yoda-1 relative to the baseline FI. Data are means ± SEM from five independent experiments. (B) B cells purified from human PBMCs with magnetic beads were transfected with either control siRNA (Control) or Piezo1-specific siRNA (Piezo1-KD) with a 4D Amaxa nucleofector. Left: PIEZO1 mRNA abundance was quantified by real-time qRT-PCR analysis and normalized to that of GAPDH mRNA. Middle: Representative histogram of total Piezo1 abundance by flow cytometry after immunofluorescence staining with an Alexa Fluor 647–labeled Piezo-specific antibody. Right: Quantification of Piezo1 abundance for B cells from four donors shown as the ratio of the Piezo1 abundance in Piezo1-KD cells relative to that in Control cells. Data are means ± SEM from four independent experiments. (C) Control and Piezo1-KD B cells were labeled with CAL520 and Ca2+ flux was measured over time by flow cytometry before and after the addition of 15 μM Yoda-1. Left: Representative Ca2+ curves after Yoda-1 treatment of the indicate cells. Right: Quantification of the percentage inhibition of the Ca2+ flux in Piezo1-KD cells as compared to that in Control cells obtained from six donors. Data are means ± SEM from six independent experiments. (D) Control and Piezo1-KD B cells were labeled with CAL520, and Ca2+ fluxes were measured over time by flow cytometry for cells treated with Yoda-1, anti-Ig, or ionomycin. Left: Representative Ca2+ kinetics plot with arrows marking the times, after baseline recording, when each stimulant was added. Right: Quantification of Ca2+ increases induced by Yoda1, soluble anti-Ig, or ionomycin were calculated as the ratio of the fold-changes in CAL520 MFI between Piezo1-KD and Control cells upon stimulation relative to the baseline MFI for B cells from nine donors. Data are means ± SEM from nine independent experiments. (E) Control and Piezo1-KD B cells were labeled with Flipper-TR and placed on glass and the lifetime decay of the dye was determined. Left: Two representative B cells showing lifetime decay. Right: Quantification of the lifetime decay of Flipper-TR for control (111 cells) and Piezo1-KD (177 cells) B cells. Scale bars, 5 μm. Data are means ± SEM from five independent experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; ns, not significant. Data in (A) to (C) were analyzed by one-sample t test with a hypothetical value of zero, data in (D) were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparisons test; and data in (E) were analyzed by two-tailed, unpaired t test with Welch’s correction.

Article Snippet: Reverse transcription and qRT-PCR qRT-PCR assays were performed with a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems), Cells-to-CT 1-Step Taqman Kit (Invitrogen, A25603), and GAPDH (NM_002046.3) and PIEZO1 (Hs00207230_m1) TaqMan Probes (Thermo Fisher).

Techniques: Purification, Labeling, Marker, Flow Cytometry, Fluorescence, Magnetic Beads, Transfection, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Inhibition, Two Tailed Test

Journal: Science signaling

Article Title: B cell responses to membrane-presented antigens require the function of the mechanosensitive cation channel Piezo1

doi: 10.1126/scisignal.abq5096

Figure Lengend Snippet: (A) Control and Piezo1-KD B cells were loaded with CAL520 and a red fluorescent dye, placed on PLB or PLB–anti-Ig, and Ca2+ fluxes were imaged at 37°C by TIRFM. Top: Representative images of color-coded Ca2+ signals normalized by cell volume for one representative cell for each condition. Bottom: Quantification of the dynamics of the Ca2+ fluxes (left) and contact areas (right) for Control B cells (13 cells) and Piezo1-KD cells (16 cells) placed on PLB and Control cells (26 cells) and Piezo1-KD cells (18 cells) placed on PLB-anti-Ig. Scale bars, 5 μm. Scale ranges are provided. Data are means ± SEM from five independent experiments. *P ≤ 0.05 and ****P ≤ 0.0001 as assessed by the ‘compareGrowthCurves’ function test. (B) Maximal Ca2+ increase (top) and maximal area (Areamax, bottom) were determined by calculating the fold-differences in the Ca2+ signal at the point of first contact with the PLB and at the point of maximal Ca2+ signal and contact area achieved during a 5-min incubation. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ns, P > 0.05. Data were analyzed by two-tailed, unpaired t test with Welch’s correction between Control and Piezo1-KD cells and by two-tailed, paired t test between PLB and PLB-anti-Ig. Data are representative of four independent experiments. (C) BCR and F-actin recruitment to the contact area imaged by TIRFM. Control and Piezo1-KD cells labeled with Alexa Fluor 488–conjugated Fab goat anti-human IgM and IgG HC were placed on either PLB or PLB-anti-Ig for the indicted times. Fixed cells were stained with Phalloidin–Alexa Fluor 647 and imaged by TIRFM. Top: Representative images of BCR and F-actin recruited to the contact area at the indicated times for Control and Piezo1-KD cells. Bottom: Quantification of BCR (left) and F-actin (right) recruitment to the contact areas from the images for >70 cells per condition from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by Kruskal-Wallis test followed by Dunn’s multiple comparison tests between PLB and PLB-anti-Ig at the indicated times, as well as between Control and Piezo1-KD cells; ns, P > 0.05. (D) Control and Piezo1-KD cells were cultured for 24 hours, labeled with the cell proliferation dye, eF450, and then cultured for 5 days in chambers with PLB or PLB–anti-Ig in the absence or presence of TH medium containing anti-CD40 antibodies, IL-21, and IL-4. Left: Representative histogram of proliferation dye for each stimulation condition in Control and Piezo1-KD cells. Right: Quantification of the percentage of proliferating cells (symbol and line graph with matched donor) and the ratio of the percentage proliferation in the Piezo1-KD cells relative to that in Control cells in the same donor. Data are means ± SEM from four donors in three independent experiments. *P ≤ 0.05 as tested by one-sample t test with a theoretical mean of zero.

Article Snippet: Reverse transcription and qRT-PCR qRT-PCR assays were performed with a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems), Cells-to-CT 1-Step Taqman Kit (Invitrogen, A25603), and GAPDH (NM_002046.3) and PIEZO1 (Hs00207230_m1) TaqMan Probes (Thermo Fisher).

Techniques: Control, Incubation, Two Tailed Test, Labeling, Staining, Comparison, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: NLRP3-Mediated Piezo1 Upregulation in ACC Inhibitory Parvalbumin-Expressing Interneurons Is Involved in Pain Processing after Peripheral Nerve Injury

doi: 10.3390/ijms232113035

Figure Lengend Snippet: SNI induces Piezo1 overexpression in ACC neurons. ( A ) SNI-induced bilateral expression of Piezo1 in the ACC. Left, Cg1 and Cg2 tissues were used for Western blotting. Coronal dissection was performed 3.0–0.5 mm anterior to the Bregma. A representative Western blot of Piezo1 expression in the bilateral ACC is shown in the middle. The protein quantification results from Western blotting are shown on the right. Significant differences in Piezo1 expression were observed on both sides of the ACC 7 d after SNI. * p -value < 0.05, ** p -value < 0.01 versus the sham group (two-way ANOVA). ( B – D ) Representative double immunofluorescence staining showing the overlap (yellow) of Piezo1-IR (red) with neuronal marker NeuN (green), but not with microglial marker Iba1 (green) and astrocyte marker GFAP (green), 7 d after SNI. The arrowhead in ( C ) indicates Iba1-IR surrounding or partially overlapping Piezo1-IR. Scale bar = 50 μm. The fluorescence intensity curves for red and green signals in the boxed areas are shown on the right side for each group.

Article Snippet: Incubation with anti-Piezo1 antibody (1:100, Alomone, Jerusalem, Israel) plus either anti-Parvalbumin antibody (1:500, NOVUS, Littleton, CO, USA), anti-CaMKⅡ recombinant rabbit monoclonal antibody (1:200, HuaBio, Zhejiang, China), anti-CD68 antibody (1:200; Abcam, Cambridge, UK) or anti-NLRP3 antibody (1:100, Affinity, Cincinnati, OH, USA) was performed overnight at 4 °C.

Techniques: Over Expression, Expressing, Western Blot, Dissection, Double Immunofluorescence Staining, Marker, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: NLRP3-Mediated Piezo1 Upregulation in ACC Inhibitory Parvalbumin-Expressing Interneurons Is Involved in Pain Processing after Peripheral Nerve Injury

doi: 10.3390/ijms232113035

Figure Lengend Snippet: SNI-induced Piezo1 is preferentially expressed in ACC PV-INs. ( A ) Representative double staining image showing the overlap (white arrows) of Piezo1-IR (red) with PV (inhibitory interneuron marker, green, below), but not with CaMK II (glutamate neuronal marker, green, top), 7 d after SNI. The color of co-localization is yellow. Blue fluorescence is from DAPI, a nuclear counterstain. Scale bar = 100 μm. ( B ) Quantitative analysis of Piezo1-IR neurons co-localized with PV-IR and CaMKⅡ-IR neurons.

Article Snippet: Incubation with anti-Piezo1 antibody (1:100, Alomone, Jerusalem, Israel) plus either anti-Parvalbumin antibody (1:500, NOVUS, Littleton, CO, USA), anti-CaMKⅡ recombinant rabbit monoclonal antibody (1:200, HuaBio, Zhejiang, China), anti-CD68 antibody (1:200; Abcam, Cambridge, UK) or anti-NLRP3 antibody (1:100, Affinity, Cincinnati, OH, USA) was performed overnight at 4 °C.

Techniques: Double Staining, Marker, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: NLRP3-Mediated Piezo1 Upregulation in ACC Inhibitory Parvalbumin-Expressing Interneurons Is Involved in Pain Processing after Peripheral Nerve Injury

doi: 10.3390/ijms232113035

Figure Lengend Snippet: The effect of recombinant rat TNF-α (rrTNF) on the expression levels of Piezo1 and NLRP3 in cellular experiments. ( A ) A representative Western blot showing Piezo1 and NLRP3 protein levels after the treatment of BV2 cells with different doses of rrTNF (0, 1, 5, and 10 ng/mL) is presented on the left. The protein quantifications are shown on the right. ** p -value < 0.01, *** p -value < 0.001 versus the control (0 ng/mL) group (two-way ANOVA). ( B ) Left: Representative double staining showing changes in the immunofluorescence intensities of caspase-1 (green) and IL-1β (red) following treatment of cultured BV2 cells with different doses of rrTNF (0, 1, 5, and 10 ng/mL) and the effect of the NLRP3 inhibitor MCC950 on the rrTNF-mediated induction of caspase-1 (green) and IL-1β in BV2 cells. Blue fluorescence is from DAPI. Scale bar = 50 μm. Right: Quantitative analysis of the fluorescence intensities of caspase-1 and IL-1β in the different groups. * p -value < 0.05, ** p -value < 0.01 (one-way ANOVA). ( C ) Representative Western blot showing Piezo1 and NLRP3 protein levels following the treatment of the primary ACC neuron culture with rrTNF (5 ng/mL) and the effect of the Piezo1 inhibitor GsMTx4 on these rrTNF-mediated inductions are shown on the top. The quantification results are presented below. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001 versus the control (one-way ANOVA).

Article Snippet: Incubation with anti-Piezo1 antibody (1:100, Alomone, Jerusalem, Israel) plus either anti-Parvalbumin antibody (1:500, NOVUS, Littleton, CO, USA), anti-CaMKⅡ recombinant rabbit monoclonal antibody (1:200, HuaBio, Zhejiang, China), anti-CD68 antibody (1:200; Abcam, Cambridge, UK) or anti-NLRP3 antibody (1:100, Affinity, Cincinnati, OH, USA) was performed overnight at 4 °C.

Techniques: Recombinant, Expressing, Western Blot, Double Staining, Immunofluorescence, Cell Culture, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: NLRP3-Mediated Piezo1 Upregulation in ACC Inhibitory Parvalbumin-Expressing Interneurons Is Involved in Pain Processing after Peripheral Nerve Injury

doi: 10.3390/ijms232113035

Figure Lengend Snippet: The effect of NLRP3 inhibition on SNI-induced Piezo1 upregulation and pain behavior. ( A ) Representative double staining showing the overlap of NLRP3-IR (red) with NeuN, Iba1 and GFAP (green), 7 d after SNI. NLRP3-IR (green) also colocalized with Piezo1-IR neurons (red). White arrow shows the co-localization (yellow). Scale bar = 50 μm. ( B ) Representative double immunofluorescence staining image showing the encapsulation of PV-IR by GSDMD-IR, a pore-forming protein downstream to NLRP3 inflammasome. Scale bar = 50 μm. Blue fluorescence in (A and B) is from DAPI. ( C ) Representative Western blot showing the effect of the NLRP3 inhibitor MCC950 (10 mg/kg i.p., once a day for 7 days) on the Piezo1 protein level in the bilateral ACC (top). Protein quantification results (bottom). * p -value < 0.05, ** p -value < 0.01 (two-way ANOVA). ( D ) Changes in ipsilateral paw withdrawal thresholds in sham, SNI + PBS, and SNI + MCC950 group. Significant differences in the threshold were observed in SNI + PBS rats but not SNI + MCC950 rats compared with sham rats on PO days 5, 11 and 15 ( n = 6). * p -value < 0.05 versus PO day −1 (Dunn’s multiple comparisons test) or # p -value < 0.05, ## p -value < 0.01 versus the sham group (multiple t test).

Article Snippet: Incubation with anti-Piezo1 antibody (1:100, Alomone, Jerusalem, Israel) plus either anti-Parvalbumin antibody (1:500, NOVUS, Littleton, CO, USA), anti-CaMKⅡ recombinant rabbit monoclonal antibody (1:200, HuaBio, Zhejiang, China), anti-CD68 antibody (1:200; Abcam, Cambridge, UK) or anti-NLRP3 antibody (1:100, Affinity, Cincinnati, OH, USA) was performed overnight at 4 °C.

Techniques: Inhibition, Double Staining, Double Immunofluorescence Staining, Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration *

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Relative mRNA levels from D54-MG glioblastoma cells and primary human astrocytes. This figure shows average relative mRNA expression for different ENaC/Deg subunits proportional to 18 S in D54-MG glioblastoma cells and primary human astrocytes. A significantly higher expression for ASIC1 (A), αENaC (B), and γENaC (C) is seen in D54-MG cells compared with astrocytes. The results are the averages of six different samples of D54-MG, and astrocytes ran in duplicate with similar results. Error bars ± 1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration *

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Knockdown of αENaC inhibits the basal whole cell current seen in D54-MG cells. A, diagram depicting the schematic of the αENaC dominant negative constructs. B1, immunoblot showing reduced expression of αENaC protein in DN-αENaC cDNA-transfected D54-MG cells compared with untransfected D54-MG control cells. B2, quantification of the blots show a 60% reduction of αENaC protein in transfected cells compared with untransfected cells. To look for the specificity of αENaC knockdown, cell lysates from B were probed for expression of ASIC1 (C1 and C2), and no difference was found in ASIC1 expression in D54-MG cells after αENaC knockdown compared with untransfected cells; n = 3 for all the blots. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, representative traces of untransfected D54-MG cells before and after infusion of 100 μm amiloride (top panel) and after knocking down αENaC (lower panel). E, the corresponding I/V curve and average conductance at −100 mV (F) showing a significant inhibition of inward current after the addition of 100 μm amiloride or after knocking down αENaC. Control traces are the same as in Fig. 3; for αENaC knockdown, n = 5. Error bars are ±1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Knockdown, Dominant Negative Mutation, Construct, Western Blot, Expressing, Transfection, Control, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration *

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Knockdown of ASIC1 cDNA and whole cell patch clamp recording. A, diagram depicting the schematic of the ASIC1 dominant negative constructs. Representative Western blot (B1) of lysates from D54-MG glioma cells transfected with DN eGFP-ASIC1 cDNA or untransfected control D54-MG cells probed for ASIC1 and quantified (B2) showing a 50–60% inhibition in ASIC1 protein expression in ASIC1 knockdown D54-MG cells compared with untransfected cells. C1, cell lysates from B were used to look for the specificity of dominant negative mutation techniques in knocking down the protein of interest. Lysates were probed by Western blot for γENaC protein expression. No difference was found in the γENaC protein expression between ASIC1 knockdown and control D54-MG cells (C2). Total actin (B1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; C1) were used as the loading control; n = 3 for all the blots. D, representative whole-cell patch clamp records of control cells (upper panel) and those transfected with ASIC1 dominant negative cDNA for 48 h (lower panel). E, I/V curve showing significant inhibition of whole cell current after the addition of 100 μm amiloride to the bath solution or after knocking down ASIC1 in D54-MG glioma cells. F, average conductance at −100 mV for each condition as in B showing significant inhibition of inward current after the addition of 100 μm amiloride or after knocking down ASIC. Traces for D54 control n = 14, for D54 + 100 μm amiloride n = 10, for ASIC1 DN and ASIC1 DN + 100 μm amiloride n = 4. Error bars ±1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Knockdown, Patch Clamp, Dominant Negative Mutation, Construct, Western Blot, Transfection, Control, Inhibition, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration *

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: γENaC Knockdown and whole cell patch clamp recording in D54-MG glioma cells. A, diagram depicting the schematic of the γENaC dominant negative constructs. B1, immunoblot showing reduced expression of γENaC protein in DN-γENaC cDNA-transfected D54-MG cells compared with untransfected D54-MG control cells. B2, quantification of the blots showing a 60–70% reduction of γENaC protein in transfected cells compared with untransfected cells. To look for the specificity of γENaC knockdown, cell lysates from B were probed for expression of ASIC1 (C1 and C2) and αENaC (D1 and D2). Immunoblots showed similar expression of ASIC1 and αENaC in both DN-γENaC cDNA-transfected D54-MG cells and in control untransfected cells. Actin was used as loading control in all traces; n = 3 for all the blots. E, representative traces of untransfected D54-MG cells before and after infusion of 100 μm amiloride (top panel) and after knocking down γENaC (lower panel). The corresponding I/V curve (F) and average conductance at −100 mV (G) showing a significant inhibition of inward current after the addition of 100 μm amiloride or after knocking down γENaC. Control traces are the same as in Fig. 3; for γENaC knockdown, n = 6. Error bars ±1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Knockdown, Patch Clamp, Dominant Negative Mutation, Construct, Western Blot, Expressing, Transfection, Control, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration *

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: ENaC/Deg subunits interact with each other in glioma cells. A, lysate from CHO-K1 cells transfected with GFP ligated ASIC1 cDNA was immunoprecipitated (IP) with mouse anti-GFP or rabbit anti-ASIC1 antibody and blotted with mouse anti-GFP antibody. The Western blot shows that immunoprecipitating with either mouse anti-GFP or rabbit anti-ASIC1 antibody and blotting with mouse anti-GFP antibody pulled down GFP-ASIC1 at 100 kDa. Western blots of total membrane fractions (B) and plasma membrane fractions (C) isolated from D54-MG cells stably transfected with ASIC1-GFP and immunoprecipitated with mouse anti-GFP, rabbit anti-ASIC1, rabbit anti-αENaC, or rabbit anti-γENaC antibody show an interaction of ASIC1 with αENaC and γENaC in D54-MG cells. Non-immune mouse IgG immunoprecipitation and immunoprecipitation with only protein A-agarose beads showed that the anti-GFP antibody was specific. D, to rule out that ASIC1 and ENaC subunit interactions were because of a random association due to overexpression, CHO-K1 and D54-MG cells were transfected with an unrelated protein; CFP ligated CLC1. Lysates from both CHO-K1 and D54-MG cells immunoprecipitated (IP) with mouse anti-GFP antibody pulled down CFP-CLC1 upon blotting with mouse anti-GFP antibody, whereas immunoprecipitating D54-MG cell lysate with rabbit anti-ASIC1 antibody did not pull down CLC1 upon blotting with mouse anti-GFP antibody. n = 3 for all the blots.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Transfection, Immunoprecipitation, Western Blot, Membrane, Clinical Proteomics, Isolation, Stable Transfection, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration *

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Knocking down ASIC1, αENaC, or γENaC inhibits D54-MG glioma cell migration. Representative images are shown of migrated D54-MG cells transfected with eYFP (A), with 100 μm benzamil in the migration buffer (B), with ASIC1 knockdown (C), αENaC knockdown (D), or with γENaC knockdown (E). The average number of cells that had migrated per field under each conditions (F) is shown. Error bars are ±1 S.D., and the asterisk indicates p < 0.05 comparing cell migration for D54-YFP with cell migration under other conditions. n = 3 for each experiment except D54-YFP, where n = 6.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Migration, Transfection, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

doi: 10.1074/jbc.m109.072793

Figure Lengend Snippet: FIGURE 1. Fast P2X7 receptor-dependent IL-1 release in peripheral blood monocytes primed with LPS, compared with P2X7 receptor-independent IL-1 release in monocytes treated with CL075. A, mono- cytes were primed with buffer (control) or LPS (1 ng/ml) for 3 h in the presence of DMSO (vehicle control) or KN62, followed by a second treatment with buffer or 300 M BzATP. Release of IL-1 was determined by ELISA (mean S.E., n 5 separate donors). Significant differences for BzATP compared with buffer only controls (***, p 0.001) or compared with KN62 (###, p 0.001) were analyzed by one-way ANOVA and Tukey’s post test. B, peripheral blood monocytes were stimulated with buffer (control), LPS (1 ng/ml), poly(I:C) (TLR3 ligand, 25 g/ml), or CL075 (TLR7/8 ligand, 1 g/ml) for 3 h, followed by buffer (control) or BzATP treatment for 30 min. The supernatants were collected, and IL-1 release was measured by ELISA (means S.E., n 5 separate donors).SignificantdifferencesforBzATPcomparedwithbufferonly(***,p0.001)wereanalyzedbyone-way ANOVA and Tukey’s post test. C, stimulated monocyte supernatants (upper panel) and lysates (lower panel) were analyzed for IL-1 content by immunoblotting, where unprocessed pro-IL-1 is 31 kDa, and active pro- cessed IL-1 (along with recombinant IL-1 control, right lane) is 17 kDa. The immunoblots shown are repre- sentative of n 3 separate donors. Con, control.

Article Snippet: The selective P2X7 receptor antagonist 15d (36) (A438079 hydrochloride; Tocris Bioscience) was used at 10 M. The caspase-1 inhibitor YVAD (Calbiochem) was incubated at 50 M in parallel with the corresponding 0.01% (v/v) DMSO vehicle control.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

doi: 10.1074/jbc.m109.072793

Figure Lengend Snippet: FIGURE 2. Specific TLR agonists mediate P2X7 receptor-independent IL-1 release in monocytes. A and B, IL-1 release by monocytes stimulated with the TLR7/8 agonist, CL075 for 3 h in the presence or absence of 15d

Article Snippet: The selective P2X7 receptor antagonist 15d (36) (A438079 hydrochloride; Tocris Bioscience) was used at 10 M. The caspase-1 inhibitor YVAD (Calbiochem) was incubated at 50 M in parallel with the corresponding 0.01% (v/v) DMSO vehicle control.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

doi: 10.1074/jbc.m109.072793

Figure Lengend Snippet: FIGURE 3. Stimulation of monocytes for 24 h with specific TLR agonists results in P2X7 receptor-independent IL-1 release. Peripheral blood monocytes treated for 24 h with 1 ng/ml LPS (A), 1 g/ml CL075 (B), 10 g/ml

Article Snippet: The selective P2X7 receptor antagonist 15d (36) (A438079 hydrochloride; Tocris Bioscience) was used at 10 M. The caspase-1 inhibitor YVAD (Calbiochem) was incubated at 50 M in parallel with the corresponding 0.01% (v/v) DMSO vehicle control.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

doi: 10.1074/jbc.m109.072793

Figure Lengend Snippet: FIGURE 6. PS exposure is dependent upon P2X7 receptor activation but independent of IL-1 release. PS exposure was assessed by flow cytometry for monocytes labeled with annexin V-PE. For each pair of micro- graphs displayed, the corresponding flow cytometric data for the same incubation conditions are shown below. A, PS staining caused by BzATP. Monocytes were primed with buffer (control, panel (i)) or LPS (1 ng/ml, panel (ii)), for 3 h and then stimulated with buffer or BzATP for 20 min. B, PS exposure inhibition by a P2X7 receptor antagonist. Monocytes were primed with buffer (control, panel (i)) or LPS (1 ng/ml, panel (ii)) for 3 h in the presence of DMSO (vehicle control) or KN62 and then stimulated with buffer or BzATP for 20 min. C, the effect of caspase-1 inhibition on PS exposure. Monocytes were primed with buffer (control, panel (i)) or LPS (1 ng/ml, panel (ii)) for 3 h in the presence of DMSO (vehicle control) or YVAD caspase-1 inhibitor and then stimulatedwithbufferorBzATPfor20min.D,theeffectofCL075onPSexposureat3hinthepresenceofDMSO (vehicle control, panel (i)) or KN62 panel (ii). Monocytes treated with CL075 were subsequently stimulated with buffer or BzATP for 20 min in the absence or presence of KN62. The data shown are histograms taken from the same donor, which are typical of n 3–4 separate donors. Further representative flow cytometric data for corresponding monocyte treatments are shown in supplemental Figs. S1 and S2.

Article Snippet: The selective P2X7 receptor antagonist 15d (36) (A438079 hydrochloride; Tocris Bioscience) was used at 10 M. The caspase-1 inhibitor YVAD (Calbiochem) was incubated at 50 M in parallel with the corresponding 0.01% (v/v) DMSO vehicle control.

Techniques: Activation Assay, Flow Cytometry, Labeling, Incubation, Staining, Control, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

doi: 10.1074/jbc.m109.072793

Figure Lengend Snippet: FIGURE 7. ATP dependence of IL-1 release from MDMs. A–D, IL-1 was measured by ELISA from superna- tants of MDMs. The cells were treated with 0.1, 1, or 10 ng/ml LPS or 0.3 and 1 g/ml CL075 for 3 h (A) or 24 h (C), followed by a 30-min stimulus with buffer or BzATP. Release of IL-1 was only detected upon a secondary stimulusofBzATP(filledbars)comparedwithnoreleaseintheabsenceofBzATP(openbars).Theadditionofthe P2X7 receptor antagonists KN62 or 15d (A438079) significantly attenuated BzATP induced IL-1 release from MDM for pretreatment over 3 h with CL075 (B) or 24 h with 1 ng/ml LPS (D). Significant differences (***, p 0.001; *, p 0.05) for comparisons indicated analyzed by one-way ANOVA and Tukey’s post test. E, MDMs were primed with buffer (control) or LPS (1 ng/ml) for 3 h, stained with Hoechst 33342 and annexin V, Alexa Fluor 488, and then stimulated with BzATP for 20 min and imaged by confocal microscopy. The images are typical of those obtained for n 3 separate donors.

Article Snippet: The selective P2X7 receptor antagonist 15d (36) (A438079 hydrochloride; Tocris Bioscience) was used at 10 M. The caspase-1 inhibitor YVAD (Calbiochem) was incubated at 50 M in parallel with the corresponding 0.01% (v/v) DMSO vehicle control.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Staining, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: List of primary and secondary antibodies used in immunohistochemistry experiments.

Article Snippet: This regional difference was confirmed using a distinct antibody targeting amino acid residues 301–313 of the extracellular loop of the rat P2X4 receptor (Apr-024 from Alomone) ( ).

Techniques: Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p < 0.05, **p < 0.01 (Student’s t -test for paired samples) represent significant differences when compared to the effects of ATP or CTP alone, respectively.

Article Snippet: This regional difference was confirmed using a distinct antibody targeting amino acid residues 301–313 of the extracellular loop of the rat P2X4 receptor (Apr-024 from Alomone) ( ).

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p < 0.05 (Student’s t -test for paired samples) represent significant differences when compared to the effect of ATP alone.

Article Snippet: This regional difference was confirmed using a distinct antibody targeting amino acid residues 301–313 of the extracellular loop of the rat P2X4 receptor (Apr-024 from Alomone) ( ).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

Article Snippet: This regional difference was confirmed using a distinct antibody targeting amino acid residues 301–313 of the extracellular loop of the rat P2X4 receptor (Apr-024 from Alomone) ( ).

Techniques: Expressing, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

Article Snippet: This regional difference was confirmed using a distinct antibody targeting amino acid residues 301–313 of the extracellular loop of the rat P2X4 receptor (Apr-024 from Alomone) ( ).

Techniques: Activity Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 1. DEPC inhibited currents through P2X2 receptors. A, traces from an oocyte expressing wild type P2X2 receptors studied before and after a 10-min exposure to DEPC made from a freshly opened bottle and dissolved in a HEPES free (phosphate-buffered) solution. B, amplitude of currents from a series of oocytes expressing wild type P2X2 in the presence of ATP plus zinc studied as in A or exposed to the phosphate-buffered solution alone for the same time interval as the DEPC-treated oocytes. The error bars indicate the S.E.; n 9 for DEPC and 11 for controls. The currents after DEPC were significantly different from those before DEPC (asterisk), but there was no significant difference between the first and second control trials. The currents in the presence of ATP alone were reduced by DEPC to a similar extent. C, at pH 6.0, DEPC inhibition of zinc potentiation of wild type P2X2 was readily detected. As indicated by the different scale bars for the currents before and after DEPC treatment, the peak currents in response to ATP were also significantly attenuated by DEPC. The dotted line indicates the amplitude of currents expected in the presence of ATP and zinc after DEPC if zinc potentiation of the remaining receptors is unchanged. D, the H120A/H213A double mutant was un- responsive to zinc, but ATP-evoked currents through these receptors were also significantly attenuated by DEPC. The amplitude of the ATP responses in these traces was scaled to cover the same distance as the ATP alone responses in C, to emphasize the lack of zinc potentiation.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Expressing, Control, Inhibition, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 2. Effect of MTSET on oocytes expressing wild type, H120C, or H213C receptors. A, traces from an oocyte expressing wild type receptors studied before and after a 1-min exposure to 5 mM MTSET. The low ATP concentration used was 10 M. In contrast to the mutant receptors shown in B, the amplitude of the response to a maximal concentration of ATP alone (200 M, indicated by the scale bar to the right of B) was nearly identical to the amplitude of the response to ATP plus zinc. B, traces from oocytes expressing H120C or H213C studied before and after a 1-min exposure to 5 mM MTSET. The low ATP concentration used was 2 M for H120C and 5 M for H213C. These concentrations were the lowest that routinely gave currents in response to ATP alone and gave the largest zinc potentiation ratios we could measure (see C). To emphasize changes in the amplitude of the currents in response to MTSET, each trace is shown twice. The upper traces are normalized to the amplitude of the currents in each oocyte in response to 200 M ATP alone before exposure to MTSET (scale bar on the right) and demonstrate that the maximal potentiation to 20 M zinc was significantly smaller than in oocytes expressing wild type receptors. The lower traces are rescaled so that the responses to ATP alone before and after MTSET treatment span the same distance (dashed lines) and emphasize the change in the amount of zinc potentiation after MTSET treatment. The time scale is identical for all traces in A and B. C, zinc potentiation (defined as ((current in low ATP 20 M zinc)/current in low ATP) 1) is shown for a series of oocytes studied at different concentrations of ATP. The error bars indicate the S.E.; n 6 for wild type, 13 for H120C, and 16 for H213C). Additional data were collected for 200 and 500 M ATP, but these are not shown because there was no significant zinc potentiation at these saturating ATP concentrations before or after MTSET. The dashed horizontal line indicates the maximal zinc potentiation observed in wild type P2X2. The dotted vertical lines indicate the average EC10 for each construct.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Expressing, Concentration Assay, Mutagenesis, Construct

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 4. Zinc concentration-response relations for wild type, H120C-, and H213C-expressing oocytes. Wild type and H120C were studied with 2 M ATP, whereas H213C was studied with 5 M ATP. For each condition, the averages of normalized data from at least three oocytes are shown. Responses to zinc concentrations greater than 20 M were corrected for the effect of the low affinity zinc inhibition as de- scribed under “Experimental Procedures.” The error bars are the S.E. The smooth curves are drawn based on the average of the EC50 and Hill coefficients to the individual fits. The vertical dashed lines indicate the EC50 for wild type P2X2 before MTSET treatment. In these experiments the MTSET treatment was at least 50 times higher than the amount sufficient to produce a maximal change in the response. The average EC50 values of both H120C- and H213C-expressing oocytes were shifted significantly to the right after treatment with MTSET.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Concentration Assay, Expressing, Inhibition

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 6. Coexpression of mutant subunits suggests an intersubunit zinc binding site. A, predicted number of intrasubunit and intersubunit binding sites. Each diagram shows the presence of a His or an Ala at positions 120 and 213 in each of the three subunits that make up a receptor. A circle indicates each type of subunit, with the N-terminal end of each subunit (the side closer to position 120) indicated by the shorter of the two wavy lines extending from each circle. B, responses of Xenopus oocytes expressing the indicated subunits to a low concentration of ATP, the same low concentration of ATP plus 20 M zinc, and then a high concentration of ATP. In all experiments, the high ATP concentration was 200 M, which produced a maximal response in all constructs used in this study. In the traces illustrated, the low ATP concentration was 10 M for P2X2 and for the mixture of H120A H213A, 20 M for H120A and 25 M for H213A. The time calibration bar is the same for all four traces. C, potentiation to high ATP was calculated as (current in high ATP/current in low ATP) 1. Thus, a potentiation factor of 9.0 is obtained for a cell studied at its EC10. These data are taken from the same oocytes used to test zinc potentiation in panel D. Each bar is the mean S.E. for at least 17 oocytes. There was no significant difference among the four conditions tested. D, zinc potentiation was calculated as ((current in low ATP 20 M zinc)/current in low ATP) 1. Thus, a potentiation factor of 0 indicates the absence of either potentiation or inhibition. A single asterisk indicates that the mean was significantly different both from P2X2 and from coinjected oocytes. A double asterisk indicates that the mean was significantly different both from P2X2 and from oocytes injected with either H120A or H213A alone.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Mutagenesis, Binding Assay, Expressing, Concentration Assay, Produced, Construct, Inhibition, Injection

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 7. Western blot analysis indicated that concatamerized trimers remain intact. Total protein from oocytes injected with RNA for wild type P2X2 monomer, or trimer HH-HH-HH, HA-AA-AH, and AA-AA-HH was separated by SDS-PAGE under reducing conditions on a 4–12% gradient gel along with protein from uninjected oocytes. Im- munoblot analysis was performed using a polyclonal antibody directed to an extracellular epitope of the human P2X2 receptor and ECL. The position of molecular mass standards (kDa) are shown on the left. In Western blots of similar gels, trimer HA-AH-HH gave a single band of the same size as the trimers shown here.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Western Blot, Injection, SDS Page

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 8. Concatameric constructs suggest an intersubunit zinc binding site. A, predicted number of intrasubunit and intersubunit binding sites in four different concatameric receptor constructs. The format of this figure is similar to Fig. 6A, except that the concatameric constructs have only one N terminus and one C terminus. B, responses of Xenopus oocytes expressing the indicated subunits to a low concentration of ATP, the same low concentration of ATP plus 20 M zinc, and then a high concentration of ATP that produced a maximal response in all of these constructs (200 M). The concentration of ATP was selected to produce a response to ATP in the absence of zinc which was 10% of the maximal response in the same cell (see C). The low ATP concentration was 7.5 M for trimer HH-HH-HH, 5 M for HA-AH-HH, 10 M for P2X2, and 12.5 M for AA-AA-HH and HA-AA-AH. C, potentiation to high ATP was calculated as in Fig. 6. These data are taken from the same oocytes used to test zinc potentiation in D. Each bar is the mean S.E. for 7–10 oocytes. There was no significant difference among the five constructs tested. D, zinc potentiation was calculated as in Fig. 6. A single asterisk indicates that the mean was significantly different from oocytes expressing P2X2 and from oocytes expressing trimers HH-HH-HH, HA-AH-HH and HA-AA-AH. A double asterisk indicates that the mean was significantly different from oocytes expressing P2X2 and from oocytes expressing trimers HH-HH-HH, HA-AH-HH, and AA-AA-HH. A triple asterisk indicates that the mean was significantly different from oocytes expressing P2X2 and from oocytes expressing trimers HH-HH-HH, HA-AA-AH, and AA-AA-HH.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Construct, Binding Assay, Expressing, Concentration Assay, Produced

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 9. A reducing agent reversibly modulated currents in the H120C/H213C double mutant, but not in wild type P2X2 receptors. A, four sequential responses from an oocyte expressing the double mutant H120C/H213C are shown. Each of the treatments indicated by the arrows was a 5-min exposure followed by a 5-min wash with recording solution alone. The DTT concentration was 10 mM, and the H2O2 concentration was 0.1%. B, summary of data for a series of 20 oocytes expressing the H120C/H213C double mutant treated as in A. The asterisks indicate that the mean response after DTT treatment was significantly different from the response before DTT treatment and also from the response after H2O2 treatment. C, average amplitude of currents evoked by 10 M ATP before and after DTT treatment in oocytes expressing wild type P2X2 receptors. DTT had no significant effect on ATP-activated currents in oocytes expressing wild type receptors. D, the concentration- response relation for oocytes expressing the H120C/H213C double mutant under nonreducing (before DTT) and reducing (after DTT) conditions. Currents for each oocyte were normalized to the peak amplitude for that cell after DTT treatment.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Mutagenesis, Expressing, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

doi: 10.1074/jbc.m504545200

Figure Lengend Snippet: FIG. 10. Cysteines at positions His120 and His213 promote cross- linking of adjacent subunits. Western blots of proteins extracted from oocytes expressing the indicated subunits in the presence (reduc- ing) or absence (nonreducing) of -mercaptoethanol in the sample run- ning buffer. The label H120C H213C indicates proteins from oocytes that were coinjected with RNAs encoding these two different subunits. The label H120C/H213C indicates proteins from oocytes injected with a single RNA encoding this double mutant. Immunoblot analysis was performed using a polyclonal antibody directed against a C-terminal epitope of the rat P2X2 receptor and ECL. The black arrows indicate the relative migration of the monomers, dimers, and trimers on a 4–12% gradient gel. A larger amount of material from the H120C/H213C double mutants was inadvertently loaded onto the gel, producing a trimer band that was so intense that the film saturated at an exposure that clearly showed the material in the other lanes. For this reason, an exposure one-third the duration of that used for the other lanes was positioned over this lane (asterisk) using Photoshop.

Article Snippet: Blots were then probed with either a goat polyclonal antibody directed against an extracellular epitope of the human P2X2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody directed against residues 460–472 of the rat P2X2 receptor (Neuromics, Bloomington, MN) and visualized by ECL (Amersham Biosciences).

Techniques: Western Blot, Expressing, Injection, Mutagenesis, Migration

Journal: bioRxiv

Article Title: ω-Phonetoxins inhibit voltage-gated calcium Ca V 2.2 ion channel splice isoforms of dorsal root ganglia

doi: 10.1101/2023.08.29.555337

Figure Lengend Snippet: Voltage dependent actions of 500 nM PnTx3-4 (A-C) and 500 nM Phα1β (D-F) on Ca V 2.2-e37a and Ca V 2.2-e37b currents evoked by voltage steps between -60 mV and +100 mV in 5 mV increments, from a holding potential of -100 mV. Average current voltage relationships (A, B, D, E), average inhibtion (left) and inhibition of individual recordings (right) at different test potentials (C, F) are shown. Average data shown are mean ± SE. ± SE.

Article Snippet: We expressed mammalian cDNAs encoding calcium-channel e37a- Cacna1b (Addgene: 26569) and e37b-Cacna1b (Addgene: 26571) splice isoforms, together with Ca V β3 (Addgene: 26574), Ca V α 2 δ-1 (Addgene, 26575) and eGFP (Clontech.

Techniques: Inhibition

Journal: bioRxiv

Article Title: ω-Phonetoxins inhibit voltage-gated calcium Ca V 2.2 ion channel splice isoforms of dorsal root ganglia

doi: 10.1101/2023.08.29.555337

Figure Lengend Snippet: A-H) , Summary of the time course of inhibition of Ca V 2.2-e37a and Ca V 2.2-e37b currents by submaximal concentration (140 nM) of PnTx3-4 (A-D) and Phα1β (E–H). Time course of toxin inhibition of individual recordings (A, E), τ on values from single exponential fits to individual data (D, G), and percental inhibition are shown. C, D, data from each cell (open symbols) are shown together with boxplots representing the interquartile range (IQR), median, and 95% confidence intervals. G, H, data from each cell (open symbolts) are shown together mean ± SE.

Article Snippet: We expressed mammalian cDNAs encoding calcium-channel e37a- Cacna1b (Addgene: 26569) and e37b-Cacna1b (Addgene: 26571) splice isoforms, together with Ca V β3 (Addgene: 26574), Ca V α 2 δ-1 (Addgene, 26575) and eGFP (Clontech.

Techniques: Inhibition, Concentration Assay

Journal: Cell Reports

Article Title: Local Resting Ca 2+ Controls the Scale of Astroglial Ca 2+ Signals

doi: 10.1016/j.celrep.2020.02.043

Figure Lengend Snippet:

Article Snippet: In these experiments, neuronal activity was inhibited by adding the sodium channel blocker TTX (1 μM, Tocris Bioscience) to the extracellular solution.

Techniques: Recombinant, Software