Figure 5: The BK channel-dependent membrane translocation of the P2X4Rs after morphine stimulation in microglia.

(a) A scatter plot of both the ATP (3 mM)-induced currents and BK currents at +30 mV in the lamina I spinal microglia after 5 days of saline or morphine (10 mg kg⁻¹) administration (two-tailed Spearman’s test, r=0.8543, P<0.0001, n=13 for Saline, n=13 for Morphine, from four mice each). (b) Typical traces of ATP (3 mM)-induced currents in the MG6 after 24 h of morphine (10 μM) stimulation. Naloxone (Nal, 10 μM) or paxilline (Pax, 2 μM) was added 1 h prior to morphine treatment. (c) The peak amplitude of the ATP-induced inward currents at a holding potential of −60 mV (n=7/8/10/10 cells). (d) An immunoblot to examine the membrane translocation of P2X4Rs following stimulation with morphine (10 μM) for 24 h in primary microglia. The cells were exposed to morphine in the presence or absence of naloxone (Nal, 10 μM) or paxilline (Pax, 2 μM). (e,f) The P2X4R levels in the membrane fraction (e) or total lysates (f) were quantified relative to the control. The data represent the means±s.e.m. ***P=0.0005 (Control versus Morphine), 0.0003 (Morphine versus Morphine+Naloxone), 0.0001 (Morphine versus Morphine+Paxilline), F_{3, 31}=10.87, a one-way ANOVA followed by the Tukey’s post hoc test (c). *P=0.0281 (Control versus Morphine), **P=0.0032 (Control versus Morphine), 0.0010 (Morphine versus Morphine+Naloxone), F_{3, 24}=7.824, a one-way ANOVA followed by the Tukey’s post hoc test (e). **P=0.0057 (Control versus Morphine), F_{3, 24}=4.956, a one-way ANOVA followed by the Tukey’s post hoc test (f). Scale bar, 50 pA and 100 ms (b).