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: Heterologous expression of hP2X1 receptors 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: Heterologous expression of hP2X1 receptors 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: Heterologous expression of hP2X1 receptors 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: Heterologous expression of hP2X1 receptors 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: Heterologous expression of hP2X1 receptors Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD).

Techniques: Incubation, Expressing, MANN-WHITNEY

Journal: European Journal of Pharmacology

Article Title: Contribution of the intracellular C terminal domain to regulation of human P2X1 receptors for ATP by phorbol ester and Gq coupled mGlu 1α receptors

doi: 10.1016/j.ejphar.2010.11.039

Figure Lengend Snippet: PMA and mGlu 1α receptor evoked potentiation of P2X1 receptors is blocked by over-expression of the intracellular C terminus. The carboxy terminus of the P2X1 receptor was co-expressed with wild type P2X1 and mGlu 1α receptors in Xenopus oocytes . (A) Upper left panels show representative currents evoked by a maximal concentration of ATP (100 μM, indicated by bar) at control oocytes (wild type P2X1) and those following 10 min incubation with PMA (100 nM). Right upper panels show the effects of over-expression of the interacellular carboxy terminus on the effects of PMA. The bar chart shows summary data, n = 5–15. (B) Upper panels show sample traces for a given oocyte co-expressing P2X1 and mGlu 1α receptors (left) or P2X1 receptors, mGlu 1α receptors and the P2X1 receptor carboxy terminal fragment (right traces). Responses to a maximal concentration of ATP (100 μM, indicated by bar) are shown before and after the application of glutamate (100 μM). Glutamate evoked an inward calcium activated chloride current and potentiated subsequent ATP evoked responses. This potentiation was reduced by co-expression of the P2X1 receptor C terminal mini-gene. The bar chart shows a summary of the data, n = 5–7. *** P < 0.001.

Article Snippet: The carboxy terminal sequence (Leu 353 –Stop 400 ) of the human P2X1 receptor was amplified from a pcDNA 3.0 vector containing the human P2X1 receptor cDNA by PCR (Techne Genius thermocycler, BioTAQ ™ DNA polymerase, Bioline, U.K.).

Techniques: Over Expression, Concentration Assay, Incubation, Expressing

Journal: European Journal of Pharmacology

Article Title: Contribution of the intracellular C terminal domain to regulation of human P2X1 receptors for ATP by phorbol ester and Gq coupled mGlu 1α receptors

doi: 10.1016/j.ejphar.2010.11.039

Figure Lengend Snippet: Properties of individual cysteine point mutations of the C terminus. (A) ATP (100 μM, application period indicated by bar) evoked rapidly desensitising currents at wild type P2X1 receptors. Cysteine mutation had no effect at the Q365C mutant but responses were reduced in amplitude by ~ 50% for R360C and by > 98% for Y363C. (B) Peak current amplitudes of ATP (100 μM) evoked currents from wild type (WT) and cysteine mutant P2X1 receptors, * P < 0.05, *** p < 0.001. (n = 4–18). (C) Total and surface expression levels of wild type and mutant P2X1 receptors with reduced peak current amplitudes are shown.

Article Snippet: The carboxy terminal sequence (Leu 353 –Stop 400 ) of the human P2X1 receptor was amplified from a pcDNA 3.0 vector containing the human P2X1 receptor cDNA by PCR (Techne Genius thermocycler, BioTAQ ™ DNA polymerase, Bioline, U.K.).

Techniques: Mutagenesis, Expressing

Journal: European Journal of Pharmacology

Article Title: Contribution of the intracellular C terminal domain to regulation of human P2X1 receptors for ATP by phorbol ester and Gq coupled mGlu 1α receptors

doi: 10.1016/j.ejphar.2010.11.039

Figure Lengend Snippet: Effects of cysteine mutations in the carboxy terminus of P2X1 receptors on PMA potentiation. (A) Representative traces of ATP evoked currents (100 μM, application indicated by bar) from oocytes under control conditions and following treatment with PMA (100 nM) for wild type (WT) and mutants H355C, I356C and K367C. (B) Summary of the percentage change in peak current amplitude to ATP following PMA treatment for P2X1 receptor cysteine mutants (n = 4–18). (C) Effects of cysteine mutations that reduced PMA potentiation when introduced into the P2X1 receptor when introduced into the C terminal fragment. Cysteine mutations P358C, K367C and Y370C abolished the inhibitory effect of the C terminal fragment on PMA regulation. (n = 5–15).

Article Snippet: The carboxy terminal sequence (Leu 353 –Stop 400 ) of the human P2X1 receptor was amplified from a pcDNA 3.0 vector containing the human P2X1 receptor cDNA by PCR (Techne Genius thermocycler, BioTAQ ™ DNA polymerase, Bioline, U.K.).

Techniques:

Journal: European Journal of Pharmacology

Article Title: Contribution of the intracellular C terminal domain to regulation of human P2X1 receptors for ATP by phorbol ester and Gq coupled mGlu 1α receptors

doi: 10.1016/j.ejphar.2010.11.039

Figure Lengend Snippet: Model of the amino and carboxy termini of the P2X1 receptor showing residues involved in PMA regulation. The effects of cysteine mutants of residues Y16-G30 (from ) and H355-Y370 on PMA regulation are shown. Residues in back abolished PMA regulation when introduced into the P2X1 receptor and removed the inhibitory effect of the corresponding mini-gene (involved in sequestering effect of mini-gene and predicted interaction with regulatory protein). Residues in grey are those that when mutated in the P2X1 receptor abolished PMA regulation but did not reduce the inhibitory effect when introduced into the C terminal fragment (transduction effect). The transmembrane segments are shown as boxes (TM1 and TM2). Secondary structural predictions of a beta sheet in the amino terminus and an alpha helix in the carboxy terminus are shown, * indicates residues that are conserved throughout the P2X receptor family and # indicates where there is a conserved positive charge.

Article Snippet: The carboxy terminal sequence (Leu 353 –Stop 400 ) of the human P2X1 receptor was amplified from a pcDNA 3.0 vector containing the human P2X1 receptor cDNA by PCR (Techne Genius thermocycler, BioTAQ ™ DNA polymerase, Bioline, U.K.).

Techniques: Transduction