Journal: Pain

Article Title: miR-1306-3p directly activates P2X3 receptors in primary sensory neurons to induce visceral pain in rats

doi: 10.1097/j.pain.0000000000002853

Figure Lengend Snippet: MiR-1306-3p activates DRG neurons through binding to P2X3Rs. (A) P2X3R was illustrated as the electrostatic potential surfaces. (B) P2X3R antagonist (A317491) significantly reduced the current induced by miR-1306-3p. Representative profiles of the currents induced by miR-1306-3p and the mix of miR-1306-3p and A317491 (10 µM, top right). Bottom, bar graph showing the reduction of the current density (n = 7 cells, *** P < 0.001, t test). (C) P2X3R antagonist A317491 significantly blocked action potentials induced by miR-1306-3p. Top, the patterns of action potential induced by miR-1306-3p (left) or the mix of miR-1306-3p and A317491 (10 µM) (right). Bottom, bar graph statistics of action potentials (n = 5 cells, * P < 0.05, t test). (D) P2X3R antagonist A317491 significantly blocked the calcium responses induced by miR-1306-3p. Top, the peaks of calcium response induced by miR-1306-3p (left), the mix of miR-1306-3p and A317491 (middle), and KCl (right) (30 mM), respectively. Bottom, bar graph statistics of ∆F340/380 signals (n = 47 cells, *** P < 0.001, t test). (E) Top: the expression of P2X3R in ND7-23 cell line with or without transfection of various siP2X3Rs (right). Bottom: siP2X3R-3 significantly reduced the expression levels of P2X3Rs. (F) Representative profiles and bar graph of the currents induced by miR-1306-3p (left) and ATP (right) on DRG neurons after the P2X3R has interfered by intrathecal injection of siP2X3R-3 for 1 week. (G) Intrathecal injection of A317491 blocked the miR-1306-3p–induced effects. Intrathecal injection of A317491 did not alter the CRD thresholds in CON rats (n = 6 rats, ** P < 0.01, * P < 0.05, one-way ANOVA). ANOVA, analysis of variance; CRD, colorectal distension; DRG, dorsal root ganglion; KCl, potassium chloride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ATP, adenosine triphosphate.

Article Snippet: Polyvinylidene fluoride was incubated with anti-P2X3R (Alomone Labs Cat# APR-026, RRID: AB_2341052, Shanghai, China) and anti-Na+-K+-ATPase (Abcam Cat# 2047-1, RRID: AB_991679, Shanghai, China) overnight at 4°C.

Techniques: Binding Assay, Expressing, Transfection, Injection

Journal: Pain

Article Title: miR-1306-3p directly activates P2X3 receptors in primary sensory neurons to induce visceral pain in rats

doi: 10.1097/j.pain.0000000000002853

Figure Lengend Snippet: The miR-1306-3p interaction sites (R180, K315, and R52) on P2X3Rs were different from the orthosteric ATP-binding sites. (A-E) The main sites of crystal structure P2X3R bind to miR-1306-3p are R180, K259, K315, and R52, respectively (red balls). (F) The ATP-binding amino acid residues (K63, K65, F174, and R281) and the predicted miR-1306-3p–binding site (R180, K259, K315, and R52) were illustrated as cyan and yellow spheres, respectively. (G) The miR-1306-3p–induced inward currents markedly decreased in the HEK293T cells expressing rP2X3R R180A , rP2X3R R52A , rP2X3R K315A , and rP2X3R K259A . Top: traces of inward currents induced by miR-1306-3p. Bottom: bar graph showing the current density (rP2X3R, n = 25; rP2X3R R180A , n = 13 cells; rP2X3R R52A , n = 20 cells; rP2X3R K315A , n = 22 cells; rP2X3R K259A , n = 20 cells; * P < 0.05, one-way ANOVA). (H) Traces and bar graph of ATP-induced inward currents (right) in HEK293T cells expressing rP2X3R R180A+K315A+R52A , whereas miR-1306-3p was incapable of inducing currents (ATP, n = 10 cells; miR-1306-3p, n = 11 cells. **** P < 0.0001). (I) Traces and bar graph of miR-1306-3p–induced inward currents in HEK293T cells expressing rP2X3R K63A+K65A+F174A+R281A , whereas ATP was incapable of inducing currents (ATP, n = 12 cells; miR-1306-3p, n = 12 cells. **** P < 0.0001). ANOVA, analysis of variance.

Article Snippet: Polyvinylidene fluoride was incubated with anti-P2X3R (Alomone Labs Cat# APR-026, RRID: AB_2341052, Shanghai, China) and anti-Na+-K+-ATPase (Abcam Cat# 2047-1, RRID: AB_991679, Shanghai, China) overnight at 4°C.

Techniques: Binding Assay, Expressing

Journal: Pain

Article Title: miR-1306-3p directly activates P2X3 receptors in primary sensory neurons to induce visceral pain in rats

doi: 10.1097/j.pain.0000000000002853

Figure Lengend Snippet: Molecular dynamics equilibrated conformations of the miR-1306-3p–P2X3R complex. (A) Computational models of the miR-1306-3p and P2X3R (apo state) complex. Three protomers of P2X3Rs were illustrated as different shades of blue. MiR-1306-3p binding to the apo P2X3R enlarged the corresponding ion channels, as indicated by comparing the crystal structure (green, without miR-1306-3p) and computational model (blue, with miR-1306-3p binding) at the transmembrane region. (B) The pore size of apo P2X3Rs is 7.5 Å (green), and the pore size of apo P2X3R binding to miR-1306-3p is 11.8 Å (blue). (C) Traces and bar graph statistics showing that inactivation time constant of miR-1306-3p–induced inward current was larger than that of ATP in DRG neurons (ATP, n = 6; miR-1306-3p, n = 9. * P < 0.05, t test). (D) Traces and bar graph showing that the inward current density of miR-1306-3p and ATP was significantly bigger than that of ATP (n = 7, ** P < 0.01, t test). (E) Traces and bar graph showing that the inward current density induced by miR-1306-3p and ATP was bigger than that of miR-1306-3p in DRG neurons (n = 8 cells, * P < 0.05, t test). (F) Intrathecal injection of miR-1306-3p antagomir and A317491 (10 µL) significantly enhanced CRD thresholds in NCI rats (n = 6 rats for each group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05, one-way ANOVA). ANOVA, analysis of variance; CRD, colorectal distension; DRG, dorsal root ganglion.

Article Snippet: Polyvinylidene fluoride was incubated with anti-P2X3R (Alomone Labs Cat# APR-026, RRID: AB_2341052, Shanghai, China) and anti-Na+-K+-ATPase (Abcam Cat# 2047-1, RRID: AB_991679, Shanghai, China) overnight at 4°C.

Techniques: Binding Assay, Pore Size, Injection

Journal: Pain

Article Title: miR-1306-3p directly activates P2X3 receptors in primary sensory neurons to induce visceral pain in rats

doi: 10.1097/j.pain.0000000000002853

Figure Lengend Snippet: Proposed working model for the role of miR-1306-3p on P2X3R channel dynamics in chronic visceral pain. miR-1306-3p activated P2X3Rs and induced inward current on the DRG neurons, thus increasing the neuron excitability and producing chronic visceral pain. DRG, dorsal root ganglion.

Article Snippet: Polyvinylidene fluoride was incubated with anti-P2X3R (Alomone Labs Cat# APR-026, RRID: AB_2341052, Shanghai, China) and anti-Na+-K+-ATPase (Abcam Cat# 2047-1, RRID: AB_991679, Shanghai, China) overnight at 4°C.

Techniques:

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure 1 Ligand induces membrane insertion of the P2X3 receptor. (A) Surface biotinylation analysis of the P2X3 receptor. HEK293T cells expres- sing P2X3-Myc were treated with 10 mM a, b-MeATP for different durations followed by cell surface biotinylation/immunoblotting. (B) HEK293T cells expressing P2X3-Myc were treated with 10 mM a, b-MeATP for 2 min plus a 28-min vehicle incubation or for 30 min plus a 10-min vehicle incubation, and subjected to cell surface biotinylation/immunoblotting. (C) HEK293T cells expressing P2X3-Myc were pre-treated with the P2X3 receptor selective antagonist A-317491 for 1 h before a, b-MeATP stimulation and subjected to cell surface biotinylation/immunoblotting. (D) Surface biotinylation analysis of the P2X1 or P2X2 receptor. HEK293T cells expressing P2X1-Myc or P2X2-Myc were treated with 10 mM a, b-MeATP or 100 mM ATP for 30 min, respectively, and subjected to cell surface biotinylation/immunoblotting. (E) Primary cultured DRG neurons were treated with 10 mM a, b-MeATP for 30 min or pre-treated with A-317491 for 1 h before a, b-MeATP stimulation and subjected to cell surface biotinylation/immunoblotting. (F) HEK293T cells expressing P2X3-Myc were pre-treated with BFA for 30 min beforea, b-MeATP stimu- lation and subjected to cell surface biotinylation/immunoblotting. TFR and actin served as internal controls for protein loading. Shown are the mean+ SEM (n ¼ 324), *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Membrane, Western Blot, Expressing, Incubation, Cell Culture

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure 2 CaMKIIa mediates a, b-MeATP-induced membrane insertion of the P2X3 receptor. (A–C) Surface biotinylation analysis of the P2X3 re- ceptor.HEK293TcellsexpressingP2X3-Mycweretreatedwith10 mMa,b-MeATPinCa2+-containingmedium(10 mMHEPESpH7.4,150 mMNaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2) or Ca2+-free medium (the above solution without Ca2+ but containing 1 mM EGTA) (A), or pre-treated with the CaMKII inhibitor KN-93 (B) or the PKC inhibitor BIM (C) for 30 min before a, b-MeATP stimulation, and subjected to cell surface biotinylation/ immunoblotting. (D) HEK293T cells coexpressing P2X3-Myc with CaMKIIa-GFP or the kinase-dead form CaMKIIaK42M-GFP were treated with 10 mM a, b-MeATP followedby cell surface biotinylation/immunoblotting. (E) HEK293T cells coexpressing P2X3-Myc with GFPor CaMKIIa-GFPor the con- stitutively active CaMKIIaT286D-GFP were subjected to cell surface biotinylation/immunoblotting. (F) CaMKIIa expression levels in both HEK293T cells and cultured DRG neurons were detected using a specific antibody. (G) Cultured DRG neurons were pre-treated with 10 mM KN-93 before a, b-MeATP stimulation followed by cell surface biotinylation/immunoblotting. TFR and actin served as internal controls for protein loading. Shown are the mean +SEM (n ¼ 324). *P , 0.05, **P , 0.01.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Membrane, Western Blot, Expressing, Cell Culture

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure 3 a, b-MeATP-activated CaMKIIa interacts with the P2X3 receptor. (A) A representative section of adult rat DRGs labeled with selective antibodies for the P2X3 receptor (green) and CaMKIIa (red). The majority of P2X3 receptor-positive neurons express CaMKIIa (arrows), and a few are negative for CaMKIIa (arrowheads). Scale bar, 30 mm. (B2D) HEK293T cells coexpressing P2X3-Myc and CaMKIIa-GFP were treated with 10 mM a, b-MeATP for different durations (B), pre-treated with A-317491 for 1 h before a, b-MeATP stimulation (C), or treated with 10 mM a, b-MeATP in Ca2+-free medium containing EGTA (D), and CaMKIIa activation levels were evaluated by immunoblot analysis of phospho-CaMKIIa (pThr286). Actin served as a loading control. (E and F) HEK293T cells coexpressing P2X3-Myc and CaMKIIa-GFP were treated with 10 mM a, b-MeATP for different durations (F) or not (E). Interaction of the P2X3 receptor with CaMKIIa was detected by immunoprecipitation with a GFP-specific antibodyand then subjected to immunoblotting with the indicatedantibodies. Shownarethe mean+SEM (n ¼ 4). *P , 0.05, **P , 0.01. (G) A diagram showing that the N and C termini of P2X3 receptor were fused to type II membrane protein transferrin receptor 1 (N-TFR-Myc) and type I membrane protein CD8a (Myc-CD8a-C), respectively (left). Myc-CD8a, Myc-CD8a-C, TFR-Myc or N-TFR-Myc were cotrans- fected with CaMKIIa-GFP in HEK293T cells. Interaction of CaMKIIa with the N or C terminus of the P2X3 receptor was detected by immunoprecipita- tion with a GFP-specific antibody and then subjected to immunoblotting with the indicated antibodies (right). (H) Interaction of the native P2X3 receptor with CaMKIIa in DRG lysates was detected by immunoprecipitation with a CaMKIIa-specific antibody and then subjected to immunoblot- ting with the indicated antibodies.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Labeling, Activation Assay, Western Blot, Control, Immunoprecipitation, Membrane

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure4 Thr388phosphorylationoftheP2X3receptorconveysCaMKIIa-mediatedmembranedelivery.(A)InHEK293TcellscoexpressingP2X3-Myc withCaMKIIaorCaMKIIaT286D,phospho-threonine(p-Thr)levelsoftheMycantibody-precipitatedP2X3receptorwereevaluatedthroughimmuno- blotting with a specific antibody against p-Thr. (B) Online prediction of the CaMKII phosphosite motif in the C terminus of P2X3 receptors from different species. The ‘K385QST388’ is the potential CaMKIIa recognition motif, and the threonine with the asterisk (Thr388 in rat) marks the residue that is potentially phosphorylated by CaMKIIa. (C) HEK293T cells expressing P2X3-Myc or P2X3T388V-Myc were treated with 10 mM a, b-MeATP followed by cell surface biotinylation/immunoblotting. (D and E) HEK293T cells expressing P2X3-Myc, P2X3T388D-Myc or P2X3T388V-Myc alone (D) or with CaMKIIa-GFP or CaMKIIaT286D-GFP (E) were subjected to cell surface biotinylation/immunoblotting. (F) In HEK293T cells coexpressing P2X3T388V-Myc with CaMKIIa or CaMKIIaT286D, phospho-threonine (p-Thr) levels of the Myc antibody-precipitated P2X3 receptor were evaluated through immunoblotting analysis with a specific antibody against p-Thr. TFR and actin served as internal controls for protein loading. Shown are the mean +SEM (n ¼ 324). *P , 0.05, ***P , 0.001.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Phospho-proteomics, Residue, Expressing, Western Blot

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure 5 Caveolin-1 is involved in a, b-MeATP-induced membrane insertion of the P2X3 receptor. (A) Representative immunoblotting and quan- tification of endogenous caveolin-1 protein in HEK293T expressing control scramble (Scr) siRNA or caveolin-1 siRNA1-3. (B) HEK293T cells coex- pressing P2X3-Myc with scramble siRNA or caveolin-1 siRNA-2 were treated with 10 mM a, b-MeATP followed by cell surface biotinylation/ immunoblotting. (C) Caveolin-1-Flag wastransfected alone or with P2X3-Myc, P2X3T388D-Myc or P2X3T388V-Myc in HEK293T cells. Caveolin-1 inter- actionwithP2X3receptorvariantswasdetectedthroughimmunoprecipitationwithaMyc-specificantibodyandthensubjectedtoimmunoblotting with the indicated antibodies. (D) HEK293T cells coexpressing P2X3-Myc and caveolin-1-Flag were treated with 10 mM a, b-MeATP for different durations. Interaction of the P2X3 receptor with caveolin-1 was detected by immunoprecipitation with a Myc-specific antibody and then subjected to immunoblotting with the indicated antibodies. (E) Interaction between the native P2X3 receptor and caveolin-1 in DRG lysates was detected through immunoprecipitation with a P2X3-specific antibody and immunoblotting with the indicated antibodies. Actin and TFR served as internal controls for protein loading. Shown are the mean+SEM (n ¼ 324). *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Membrane, Western Blot, Expressing, Control, Immunoprecipitation

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure 6 Caveolin-1 is indispensable for a, b-MeATP-induced membrane insertion of the P2X3 receptor. (A) Myc-CD8a, Myc-CD8a-C, TFR-Myc, or N-TFR-Myc wascotransfected with caveolin-1-GFP in HEK293T cells. The interaction of caveolin-1with the N or C terminus of the P2X3receptorwas detected through immunoprecipitation with a GFP-specific antibody and immunoblotting with the indicated antibodies. (B and C) HEK293T cells expressing P2X3-Myc, P2X3△N-Myc, P2X3T388D-Myc, P2X3△N, T388D-Myc (B), or P2X3△C-Myc (C) were subjected to cell surface biotinylation/im- munoblotting. (D) A diagram mapping caveolin-1 and CaMKIIa binding regions in the N terminus of the P2X3 receptor by alanine scanning muta- genesis. Lysates from HEK293T cells coexpressing caveolin-1-GFP or CaMKIIa-GFP with N-TFR-Myc or various mutants were incubated with a GFP-specific antibodyand subjected to coimmunoprecipitationand immunoblotting with the indicatedantibodies.(EandF) HEK293T cellsexpres- sing P2X3FFTYET-AAAAAA-Myc, P2X3FFTYET-AAAAAA, T388D-Myc, P2X3-Myc, P2X3KSWTI-AAAAA-Myc, or P2X3KSWTI-AAAAA, T388D-Myc were subjected to cell surface biotinylation/immunoblotting. TFR served as an internal control for protein loading. Shown are the mean+ SEM (n ¼ 324). *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Membrane, Immunoprecipitation, Western Blot, Expressing, Binding Assay, Incubation, Control

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure7a,b-MeATPinducesthemembranecoinsertionoftheP2X3andP2X2receptors.(A)ArepresentativesectionofadultratDRGtriple-labeled with selective antibodies of P2X3 (green), P2X2 (blue), and CaMKIIa (red). The majority of P2X2 and P2X3 receptor-positive neurons express CaMKIIa (arrows). Scale bar, 30 mm. (B) HEK293T cells coexpressing P2X2-Myc and P2X3-Myc were treated with 10 mM a, b-MeATP for 30 min or pre-treated with 10 mM KN-93 for 30 min before a, b-MeATP stimulation and then subjected to cell surface biotinylation/immunoblotting. TFR served as a loading control for surface proteins. Shown are the mean+SEM (n ¼ 3). *P , 0.05 versus control cells treated with vehicle, #P , 0.05 versus cells treated with a, b-MeATP. (C) HEK293T cells coexpressing P2X2-Myc with P2X3-Myc or P2X3T388D-Myc were subjected to cell surface biotinylation/immunoblotting. Shown are the mean+ SEM (n ¼ 5). *P , 0.05 versus cells coexpressing P2X3-Myc and P2X2-Myc. (D) P2X3-Myc or P2X3T388D-Myc was cotransfected with P2X2-Myc in HEK293T cells. The interaction of P2X2-Myc with P2X3-Myc or P2X3T388D-Myc was detected by coimmunoprecipitation with a P2X3 receptor-specific antibody and then immunoblotting with the indicated anti- bodies. (E) Primary cultured DRG neurons were treated with 10 mM a, b-MeATP for 30 min and subjected to cell surface biotinylation/immuno- blotting. TFR served as an internal control for surface protein loading. Shown are the mean+ SEM (n ¼ 3). **P , 0.01. (F) HEK293T cells expressing the indicated receptors were cross-linked with the non-permeable and irreversible BS3 followed by immunoblotting to detect the homotrimeric and heterotrimeric receptors on the cell membrane.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Labeling, Western Blot, Control, Cell Culture, Expressing, Membrane

Journal: Journal of molecular cell biology

Article Title: CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor.

doi: 10.1093/jmcb/mju011

Figure Lengend Snippet: Figure 8 Phosphorylation-increased P2X3 receptors on the cell membrane are functional. (A and B) HEK293T cells expressing P2X3-Myc, P2X3T388D-Myc, or P2X3T388V-Myc were treated with 10 mM a, b-MeATP for 5 min followed by immunoblotting analysis to detect ERK activation. Shownarethemean+ SEM(n ¼ 4).*P , 0.05,**P , 0.01.(C)AschematicdiagramshowingtheATP-inducedmembranedeliveryoftheP2X3and P2X2/3 receptors. Extracellular ATP binds to and activates the P2X3 receptors present on the cell membrane in the soma and the nerve terminal of DRG neurons. Ca2+ influx via the P2X3 receptor not only phosphorylates ERK to form a ‘signaling endosome’ but also activates CaMKIIa, which binds to the N terminus of the P2X3 receptor and phosphorylates Thr388 in the C terminus. Thr388 phosphorylation of the P2X3 receptor enhances its binding to caveolin-1, leading to the promotion of forward trafficking and membrane insertion of the P2X3 receptor. CaMKIIa is dispensable in forward transport of the P2X3 receptor after Thr388 phosphorylation. Furthermore, this regulated trafficking of the P2X3 receptor also drives the membrane delivery of the assembled P2X2 receptor and enhances the P2X3 receptor-mediated response.

Article Snippet: The samples were separated on SDS–PAGE, transferred, probed with P2X3 (Neuromics), TFR (Invitrogen), actin (Chemicon), Myc (Proteintech), GFP (Roche), pCaMKIIa, CaMKIIa, caveolin-1, pERK (Santa Cruz), CaMKII (Millipore), phospho-threonine (Cell Signaling Technology), phosphoserine (Sigma), Flag (Abmart), P2X2 (Chemicon), or ERK (Abcam) antibodies and visualized with enhanced chemiluminescence (Amersham Biosciences).

Techniques: Phospho-proteomics, Membrane, Functional Assay, Expressing, Western Blot, Activation Assay, Binding Assay

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Daily i.t. injection of the selective P2X3 receptor antagonist A317491 attenuates morphine‐induced anti‐nociceptive tolerance. (A) Schematic of the experimental timeline. (B) Thermal and (C) mechanical thresholds measured daily in all groups (n = 6; *P < 0.05 vs. saline group; # P < 0.05 vs. morphine group).

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Injection

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: (A) Expression of P2X3 receptor and (B) number of P2X3 receptor‐positive cells per section per animal in DRGs increase after repeated morphine treatment. The DRGs were removed 2 h after morphine injection on day 7 (n = 6,*P < 0.05 vs. saline (S) group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Injection

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Morphine‐induced P2X3 receptor up‐regulation is co‐localized with IB4 and peripherin in the DRG. (A) Confocal images of P2X3 receptor (green) and ionized Ca2+‐binding adapter molecule 1 (Iba1) (red) immunofluorescence with no co‐localization. (B) Immunofluorescence images of P2X3 receptor (red) and IB4 (green) co‐localization in DRGs. (C) There was less co‐localization of P2X3 receptor (green) with NF200 (red) in myelinated neurons. (D) Co‐localization of P2X3 receptor (green) with peripherin (red) in unmyelinated/small‐calibre neurons. DRG samples from the 7 day morphine group (scale bars: A/B, 80 μm; C/D, 20 μm).

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Binding Assay, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Involvement of PKCε in morphine (Mor)‐induced antinociceptive tolerance and P2X3 receptor expression. (A) The phosphorylation of PKCε in the DRG increased time dependently with chronic Mor treatment. (B) The inhibitor of PKCε, ε‐V1‐2, reversed the increased P2X3 receptor expression induced by repeated Mor treatment. (C) PKCε co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the up; 20 μm for the down). (D) Mor tolerance was attenuated when Mor was administered with ε‐V1‐2 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Injection

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Epac is responsible for the chronic morphine (Mor)‐induced phosphorylation of PKCε and increase in P2X3 receptor expression. (A) The expression of Epac in the DRG increased time dependently after chronic Mor exposure. (B) The inhibitor of Epac, ESI‐09, reversed the increased P2X3 receptor expression and phosphorylation of PKCε induced by repeated morphine treatment. (C) Epac co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the left; 20 μm for the right). (D) Morphine tolerance was attenuated by co‐administration of morphine and ESI‐09 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Injection