Journal: Neuroscience

Article Title: Tonic calcium-activated chloride current sustained by ATP release and highly desensitizing human P2X1 receptors

doi: 10.1016/j.neuroscience.2019.07.025

Figure Lengend Snippet: Spontaneous transient inward currents (STICs) in Xenopus oocytes expressing hP2X1 receptors. A. Oocytes expressing hP2X1 receptors, voltage-clamped at −80 mV, showed inward currents during mechanical deformation induced by stopping (off) or reinitiating (on) the perfusion flow. B. ATP response of an oocyte expressing hP2X1 receptors. Insert, magnification of the baseline showing membrane oscillations before ATP application, and their absence after ATP perfusion, calibration bars indicates values for the recording in panel B and the insert. C. Individual STICs in two oocytes recorded at 24 and 48 hrs after injection of cRNA for the hP2X1 subunit.

Article Snippet: Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD).

Techniques: Expressing, Injection

Journal: Neuroscience

Article Title: Tonic calcium-activated chloride current sustained by ATP release and highly desensitizing human P2X1 receptors

doi: 10.1016/j.neuroscience.2019.07.025

Figure Lengend Snippet: STICs are dependent on the activation of hP2X1 receptors. A. STICs in an oocyte injected with cRNA for hP2X1 in resting conditions and voltage clamped at −80 mV. B. Distribution of STICs amplitude in 10 oocytes. Median = 9.5 nA. Insert, amplification of a STIC from the oocyte shown in A (arrow). C. Blocking of STICs and reduction of the holding current (open conductance) by NF023, a P2X1 antagonist. D. Apyrase, which degrades ATP, also reduced the STICs amplitude and the holding current. E. Scanning Electron Microscope (SEM) photograph of a fixed oocyte that had STICs and was completely devoid of follicular cells on its surface. Xenopus oocytes were processed as previously described (Miledi and Woodward, 1989) and photographed using a FEI Quanta 3D FEG Dual Beam (SEM/FIB) microscope at 10KV.

Article Snippet: Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD).

Techniques: Activation Assay, Injection, Amplification, Blocking Assay, Microscopy

Journal: Neuroscience

Article Title: Tonic calcium-activated chloride current sustained by ATP release and highly desensitizing human P2X1 receptors

doi: 10.1016/j.neuroscience.2019.07.025

Figure Lengend Snippet: Effects of Ba2+ substitution on STICs and tonic current. A. Membrane oscillations of an oocyte expressing hP2X1 receptors at rest in normal Ringer’s solution, in a solution where Ba2+ replaced Ca2+, and during the application of NF023. The oocyte was voltage clamped at −80 mV. Notice that returning to normal Ringer produced a rebound of STICs and tonic current. The current resistant to NF023 was used as the baseline (zero) for current measurements B. Cumulative distribution of the inward current sensitive to Ba2+ and NF023 in 60 seconds epochs. C. Normalized current, at the 0.5 value of the cumulative distribution, for the different conditions shown in A (n = 21 oocytes). **, p<0.0005; *** p<0.0001, Oneway ANOVA followed by multiple comparisons vs Ringer’s solution control using Dunnett’s method.

Article Snippet: Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD).

Techniques: Expressing, Produced

Journal: Neuroscience

Article Title: Tonic calcium-activated chloride current sustained by ATP release and highly desensitizing human P2X1 receptors

doi: 10.1016/j.neuroscience.2019.07.025

Figure Lengend Snippet: Effects of BAPTA-AM on STICs and tonic current. A. The efficacy of 50 μM BAPTA-AM to chelate intracellular Ca2+ was monitored by the complete blockade of Ca2+ oscillations induced by 1:1000 RS. B. Membrane oscillations of an unstimulated oocyte expressing hP2X1 receptors and incubated overnight in 50 μM BAPTA-AM. Notice that presence of STICs in Ringer with Ca2+ and the strong reduction in amplitude when Ba2+replaced Ca2+. The current resistant to NF023 was used as the baseline (zero) for current measurements. C. Cumulative distribution of the inward current sensitive to Ba2+ and NF023 in 60 seconds epochs. D. Normalized current, at the 0.5 value of the cumulative distribution, for the different conditions shown in A (n = 10 oocytes). *, p<0.01; ** p<0.05, Oneway ANOVA followed by multiple comparisons vs Ringer’s solution control using Dunnett’s method.

Article Snippet: Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD).

Techniques: Expressing, Incubation

Journal: Neuroscience

Article Title: Tonic calcium-activated chloride current sustained by ATP release and highly desensitizing human P2X1 receptors

doi: 10.1016/j.neuroscience.2019.07.025

Figure Lengend Snippet: Effects of brefeldin incubation on STICs. A. Representative current trace of oocytes expressing hP2X1 receptors after overnight incubation in 50 μM BAPTA-AM and 3 hours treatment in 20 μM brefeldin. In this condition oocytes did not show STICs, before, during, or after perfusion with Ba2+ solution. A persistent tonic current is still present as well as the current rebound after Ba2+ (arrow). B. Comparison between oocytes treated with brefeldin, with and without incubation with BAPTA-AM; ** p<0.001, Mann-Whitney U test (n = 5; each group)

Article Snippet: Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD).

Techniques: Incubation, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A-C) P2X7 expression analysed by western blotting of macrophages isolated from control, EROS -/- (A) and gp91 phox -/- mice (B) and of control PLB985 cells and an EROS knock-out clone (C). (D-E) P2X7 expression in RAW264.7 cells overexpressing a FLAG-tagged EROS vector (D) and in HEK293 cells transiently expressing the specified constructs (E). (F-G) Interaction between EROS and P2X7 probed by immunoprecipitation of EROS from RAW264.7 EROS-FLAG macrophages followed by immunoblot for P2X7 (F) and by NanoBIT assay in live HEK293 cells expressing the LgBIT-fused EROS vector with a SmBIT-fused P2X7 vector (G). (H) P2X1 expression in macrophages isolated from EROS -/- mice compared to control. n= 5 biological replicates. (I) P2X1 abundance upon co-transfection with EROS construct in HEK293 cells. n= representative of 3 independent experiments. See also Figure S3.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Expressing, Western Blot, Isolation, Knock-Out, Plasmid Preparation, Construct, Immunoprecipitation, Cotransfection