Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function
Fig 1
Co-expression of hP2X7R-Gln460Arg with hP2X7R-WT diminishes normal receptor function.
(A) Increase of intracellular calcium of stably transfected HEK293 cells was measured following BzATP application (50 μM) (repeated measures ANOVA, P < 0.01 hP2X7R-WT and hP2X7R-Gln460Arg versus HEK293; n = 4). For each cell line, nine individual clones were analyzed (B) Left: representative whole-cell measurements out of four independent experiments by whole-cell patch clamp analysis. Right: Quantification of inward currents elicited by BzATP (One-way ANOVA with Scheffé’s test, ns = non-significant versus hP2X7R-WT; n = 4) (C) Increase of intracellular calcium of HEK293 cells stably transfected with hP2X7R-WT (9 clones) and stably double transfected with hP2X7R-WT + hP2X7R-Gln460Arg (10 clones) was measured (repeated measures ANOVA, P < 0.01 hP2X7R-WT + Gln460Arg versus hP2X7R-WT; n = 4). (D) Left: representative whole-cell measurements by whole-cell patch clamp analysis. Right: Quantification of inward currents elicited by BzATP (One-way ANOVA with Scheffé’s test, *P < 0.05 versus hP2X7R-WT; n = 4) (E) BzATP (50 μM)-induced activation of p-ERK 1/2 in HEK293 cells expressing P2X7R variants. Each value of pERK1/2 was normalized to total ERK1/2. Results are expressed as the percentage of maximum pERK1/2 obtained at 2 minutes of stimulation in hP2X7R-WT cells ± s.e.m. from 3 independent experiments. One-way ANOVA, * P < 0.05 versus hP2X7R-WT and versus hP2X7R-Gln460Arg at the same time points. Bottom panels show WBs of pERK1/2 and total ERK1/2 from a representative experiment. (+): Fetal calf serum 10% treatment for 10 min, positive control for p-ERK 1/2 activation. Inset: Quantification and representative example showing WB detection of hP2X7R variants in parental HEK293 cells and analyzed stable clones.