Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: P2X7 receptor-mediated dye uptake is enhanced by depletion of plasma membrane cholesterol and reduced by loading of cholesterol. A , example confocal images captured during 245-s time series to measure dye uptake. Time of image capture, in seconds, is shown. Ethidium bromide was added at 20 s, and 300 μ m BzATP was added at 45 s. The increase in fluorescence as a result of ethidium uptake ( red ) into P2X7-expressing cells ( green ) is apparent. B , human P2X7-mediated dye uptake was slower than mouse P2X7-mediated dye uptake ( n = 3 coverslips, ∼10–30 cells per coverslip, p < 0.001), normalized to human P2X7. C , measurements of cholesterol ( chol ) in TSA 201 cells. A 15-min preincubation with 5 or 15 m m MCD reduced cholesterol, 5 m m αCD for 15 min had no effect, and 100 μg/ml water soluble cholesterol for 30 min increased cholesterol ∼2-fold ( n = 3, *, p < 0.01, **, p < 0.001; ns , not significant). D , time course of human P2X7-mediated dye uptake, normalized to control. Uptake was enhanced by 5 m m MCD and inhibited by 100 μg/ml cholesterol ( n = 3–4, p < 0.05). E , 5 m m αCD has no effect on human P2X7-mediated dye uptake, normalized to the untreated condition ( n = 3, p > 0.05). F , rate of P2X7-mediated dye uptake 10–50 s after agonist application, normalized to control. Human P2X7 dye uptake was increased ∼5-fold by 5 m m MCD and almost abolished by 100 μg/ml cholesterol ( n = 66 alone, n = 64 MCD and n = 6 cholesterol, **, p < 0.001). Mouse P2X7 dye uptake was increased ∼2.2-fold by MCD and reduced by cholesterol ( n = 88 alone, n = 50 MCD and n = 4 cholesterol, **, p < 0.001). G , concentration-response curves for mouse P2X7 receptor-mediated dye uptake, with and without pretreatment with 5 m m MCD. Responses were normalized to the rate of increase in fluorescence in untreated cells stimulated with 300 μ m BzATP. H , time course of dye uptake mediated by the more active mouse P2X7k variant, stimulated with 30 μ m BzATP, normalized to the untreated condition ( n = 3, p < 0.05 for MCD, p < 0.001 for cholesterol). I , rat P2X7 dye uptake was enhanced by 5 m m MCD, but not significantly ( p = 0.07), and inhibited by 100 μg/ml cholesterol ( n = 3, p < 0.05), normalized to the untreated condition. J , in mouse bone marrow-derived macrophages, dye uptake via endogenous P2X7 was potentiated by 5 m m MCD and reduced by 100 μg/ml cholesterol ( n = 4, p < 0.001 for MCD, p < 0.01 for cholesterol), normalized to the untreated condition.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Fluorescence, Expressing, Concentration Assay, Variant Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The enhancement in P2X7 receptor-mediated dye uptake by MCD treatment is independent of pannexin-1 and cytoskeletal changes. A , neither carbenoxolone (+ carb ; 30 μ m ) nor pretreatment with the caspase inhibitor zVAD-FMK (+ zVAD ; 5 μ m for 1 h) reduced human P2X7-mediated ethidium uptake following MCD treatment. ( n = 6, p > 0.05). B , preincubation with (−)-blebbistatin (+ bleb ; 100 μ m for 1 h), an inhibitor of non-muscle myosin 2, had no effect on the rate of mouse P2X7-mediated dye uptake, with or without MCD pretreatment ( n = 12, p > 0.05). C , the actin-stabilizing agent jasplakinolide (+ jasp ; 30 n m for 1 h) had no effect on human P2X7-mediated dye uptake with or without MCD treatment ( n = 4–8, p > 0.05). D , latrunculin-A (+ lat ; 5 μ m for 30 min), an inhibitor of F-actin polymerization, had no effect on the initial rate of mouse P2X7-mediated dye uptake, with or without MCD treatment ( n = 3–6, p > 0.05).

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: Acute changes in cholesterol affect P2X7 whole-cell current amplitude, sensitization and NMDG permeability. A , example traces of whole-cell patch clamp recordings from HEK 293 cells expressing the mouse P2X7 receptor and enhanced GFP, stimulated with repeated 5-s applications of 300 μ m BzATP followed by a 1-min wash. chol , cholesterol. B , mean current densities after one or more applications of BzATP, in untreated cells or cells pretreated with 5 m m αCD, 5 m m MCD, or 100 μg/ml cholesterol ( n = 3–18 cells, *, p < 0.01, **, p < 0.001; ns , not significant). pF , picofarads. C , example traces of whole-cell recordings from cells expressing the human P2X7 receptor, stimulated with repeated 5-s applications of 300 μ m BzATP followed by a 1-min wash. D , mean current density upon first agonist application, with and without 5 m m MCD pretreatment ( n = 18 cells, **, p < 0.001). E , -fold change in current density from first agonist application to last application (>10), where each point represents one cell. The solid line shows mean -fold change, and the dotted line is at 1. F–H , mouse P2X7 receptor currents in the NMDG + extracellular solution, stimulated with 300 μ m BzATP for 40 s. Mean peak current densities and the 10–90% rise time are shown ( n = 7–9, *, p < 0.01). I , TSA 201 cells expressing mouse P2X7 or human P2X7 receptors were untreated or treated with 5 m m MCD for 15 min and then surface-labeled with sulfo-NHS-LC-biotin. For mouse P2X7, 3 wells of a 6-well plate of cells were then solubilized in 350 μl of solubilization buffer, whereas for human P2X7, a T75 flask of cells was solubilized in 1 ml. The Western blot shows the total lysate ( T ) and surface-biotinylated sample ( B ) blotted for P2X7 and β-actin. MCD treatment did not affect the proportion of P2X7 receptors at the surface.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Permeability, Patch Clamp, Expressing, Labeling, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in gray , P2X1 aa20–23 and equivalent residues in P2X2 underlined , and the P2X7 residues Lys 17 and Val 18 in red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7. C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT: n = 14 alone, n = 11 MCD, n = 3 cholesterol; mutants: n = 12 alone, n = 11 MCD, n = 3 cholesterol; *, p < 0.05, **, p < 0.001; ns , not significant). D , whole-cell patch clamp recordings of human P2X7 K17R/V18M receptor ± 5 m m MCD pretreatment, stimulated with repeated 5-s applications of 300 μ m BzATP followed by a 1-min wash. E , mean peak current density upon first application of BzATP. MCD pretreatment potentiates initial current density ( n = 10–17 cells, **, p < 0.001), but the extent of potentiation is less than the wild type receptor ( p < 0.05, two-way analysis of variance). pF , picofarads.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Activity Assay, Sequencing, Mutagenesis, Relative Rate, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: Cholesterol-sensing within the proximal C terminus of the P2X7 receptor. A , amino acid sequence of the human P2X7 N terminus and TMD1, and the end of the TMD2 and proximal C terminus. The CRAC motifs are underlined , with constituent residues in bold and central tyrosines in red. Arrows indicate the amino acids reported to interact with phosphatidylinositol 4,5-bisphosphate , and the cluster of hydrophobic residues downstream of these is in bold/blue . The 18-amino acid cysteine-rich region is highlighted in gray , and its cysteines are in bold/green. B , bar graph of the relative rates of dye uptake ± 5 m m MCD for P2X7 Tyr to Phe mutants normalized to the untreated WT P2X7 (WT: alone n = 16, MCD n = 5; mutants: n = 3–6; *, p < 0.01, **, p < 0.001; ns , not significant). C , cells expressing P2X7 WT or P2X7 mutants were surface-labeled with sulfo-NHS-LC-biotin. The Western blot shows the total lysate ( T ) and surface-biotinylated sample ( B ) blotted for P2X7 and β-actin. The Y51F mutation inhibited surface and total expression of the receptor. D , bar graph of the relative rates of dye uptake ± 5 m m MCD, for hydrophobic to alanine mutants normalized to the untreated WT P2X7 (WT: n = 9; mutants: n = 3–6; *, p < 0.05, **, p < 0.001). E , cells expressing P2X7 WT or P2X7 mutants were surface-labeled with sulfo-NHS-LC-biotin. The Western blot shows the total lysate ( T ) and surface-biotinylated sample ( B ) blotted for P2X7 and β-actin. Surface expression was inhibited by V401A, F403A, and V404A mutations.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Sequencing, Expressing, Labeling, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: Bromopalmitate pretreatment inhibits surface expression of the P2X7 receptor but does not abolish sensitivity to MCD. A and B , time course of dye uptake by mouse P2X7 receptor ( A ) and human P2X7 receptor ( B ) after 16 h of pretreatment with 100 μ m BrP or DMSO (control). P2X7 receptor-mediated dye uptake in BrP-treated cells was significantly potentiated by 5 m m MCD ( n = 4 for mouse, n = 3 for human, p < 0.05). C , HeLa cells expressing mouse P2X7-FLAG receptors were immunostained by live labeling with anti-FLAG antibody. BrP reduced the surface expression of mouse P2X7-FLAG, but MCD treatment did not ( scale bar = 20 μm, n = 32–40 cells per condition, **, p < 0.001; ns , not significant). D , Western blot of TSA 201 cells expressing mouse P2X7 shows that pretreatment with BrP reduces total expression of the receptor.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Expressing, Labeling, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The cysteine-rich region within the proximal C terminus of P2X7 is important for normal pore dilation. A , time course of dye uptake mediated by P2X7 Δ18aa ± 5 m m MCD normalized to the untreated WT P2X7. B , relative rates of dye uptake mediated by P2X7 Δ18aa ± MCD, normalized to untreated WT P2X7 (WT: n = 6 alone, n = 2 MCD; mutants: n = 7 alone, n = 4 MCD; *, p < 0.05). C , Western blot shows that total ( T ) and surface-biotinylated ( B ) expression of P2X7 Δ18aa was similar to WT P2X7 ( panel B ). D , example whole-cell current recording for P2X7 Δ18aa stimulated with repeated 5 s applications of 300 μ m BzATP. Currents had slow activation kinetics, exhibited run down, and were flickery. E , the mean peak current density for the first agonist application appeared to be reduced by MCD treatment, although this was not significant (alone n = 13, MCD n = 8, p > 0.05). F , bar graph of the relative rates of dye uptake ± 5 m m MCD for P2X7 Cys to Ala mutants, normalized to untreated WT P2X7. (WT: alone n = 8, MCD n = 11; Cys to Ala mutants: n = 3–6; *, p < 0.05, **, p < 0.001; ns , not significant). G , Western blot shows that surface expression of C363A and C373A mutants was similar to WT, whereas C371A and C377A mutants had reduced surface expression. H , whole-cell recording of P2X7 C363A receptor currents, stimulated with repeated 5-s applications of 300 μ m BzATP. I , P2X7 C363A mean peak current density upon first application of BzATP was potentiated by 5 m m MCD pretreatment ( n = 5, *, p < 0.05).

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Western Blot, Expressing, Activation Assay