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Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization.

Robinson LE, Shridar M, Smith P, Murrell-Lagnado RD - J. Biol. Chem. (2014)

Bottom Line: This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes.Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating.These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.

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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 μm 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 mm αCD, 5 mm 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 μm BzATP followed by a 1-min wash. D, mean current density upon first agonist application, with and without 5 mm 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 μm 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 mm 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.
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Figure 3: 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 μm 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 mm αCD, 5 mm 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 μm BzATP followed by a 1-min wash. D, mean current density upon first agonist application, with and without 5 mm 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 μm 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 mm 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.

Mentions: Whole-cell currents mediated by the mouse and human P2X7 receptors differ in the degree and rate at which they sensitize in response to exposure to agonist, with human P2X7 showing a more pronounced and slower rate of sensitization (13). We tested the effects of depleting or loading cholesterol on currents activated by repetitive 5-s applications of 300 μm BzATP at both receptors. The mouse P2X7 receptor gave reproducible responses that were strongly inhibited by pretreatment with MCD loaded with cholesterol (Fig. 3, A and B). Treatment with MCD alone dramatically enhanced the amplitude of the current evoked by the first application of agonist and, although subsequent responses were also of larger amplitude, they showed some desensitization, unlike the untreated cells. For the human P2X7 receptor, preincubation with MCD also caused a large increase in the amplitude of the initial currents (Fig. 3, C and D). Under control conditions, recordings showed considerable variability in the degree of run-up (Fig. 3E). Approximately 40% showed a profound run-up after >10 applications of BzATP; for this group, the mean increase was ∼6.1-fold. Following MCD treatment, initial currents were already a similar magnitude to that seen after run-up of untreated cells, and only two recordings subsequently showed a modest run-up. Thus, for both mouse and human P2X7, the effect of MCD is to sensitize the receptor to the initial application of agonist.


Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization.

Robinson LE, Shridar M, Smith P, Murrell-Lagnado RD - J. Biol. Chem. (2014)

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 μm 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 mm αCD, 5 mm 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 μm BzATP followed by a 1-min wash. D, mean current density upon first agonist application, with and without 5 mm 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 μm 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 mm 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.
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Related In: Results  -  Collection

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Figure 3: 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 μm 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 mm αCD, 5 mm 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 μm BzATP followed by a 1-min wash. D, mean current density upon first agonist application, with and without 5 mm 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 μm 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 mm 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.
Mentions: Whole-cell currents mediated by the mouse and human P2X7 receptors differ in the degree and rate at which they sensitize in response to exposure to agonist, with human P2X7 showing a more pronounced and slower rate of sensitization (13). We tested the effects of depleting or loading cholesterol on currents activated by repetitive 5-s applications of 300 μm BzATP at both receptors. The mouse P2X7 receptor gave reproducible responses that were strongly inhibited by pretreatment with MCD loaded with cholesterol (Fig. 3, A and B). Treatment with MCD alone dramatically enhanced the amplitude of the current evoked by the first application of agonist and, although subsequent responses were also of larger amplitude, they showed some desensitization, unlike the untreated cells. For the human P2X7 receptor, preincubation with MCD also caused a large increase in the amplitude of the initial currents (Fig. 3, C and D). Under control conditions, recordings showed considerable variability in the degree of run-up (Fig. 3E). Approximately 40% showed a profound run-up after >10 applications of BzATP; for this group, the mean increase was ∼6.1-fold. Following MCD treatment, initial currents were already a similar magnitude to that seen after run-up of untreated cells, and only two recordings subsequently showed a modest run-up. Thus, for both mouse and human P2X7, the effect of MCD is to sensitize the receptor to the initial application of agonist.

Bottom Line: This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes.Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating.These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.

Show MeSH
Related in: MedlinePlus