Figure 6

Cdk5 and calcineurin modulate P2X ₃ receptors of trigeminal neurons. A, Example of western immunoblot using anti-Cdk5 antibodies on membrane extracts from WT and KI neurons. Total Cdk5 in the soluble fraction is also shown. Actin immunoblotting ensures equal loading between WT and KI fractions (bottom row). Histograms (right) quantify average data (n = 4, p = 0.04) of membrane Cdk5. B, Example of co-immunoprecipitation experiments between P2X₃ receptors and Cdk5 in WT and KI neurons showing decreased detection of Cdk5 in P2X₃ immunopurified samples from KI neurons. Histograms (right) quantify lower Cdk5 signal from KI neurons (n = 3, p = 0.03). C, Example of current traces recorded from WT and KI trigeminal neurons evoked by α,β-meATP (10 μ M, 2-s application) in control and after incubation with FK-506 (5 μ M, 30 min). Histograms (right) quantify the effect of FK-506 on the P2X₃ receptor-mediated currents (n = 18, *p = 0.035 in WT neurons and n = 20, *p = 0.027 in KI neurons). Data are normalized to the signals from WT neurons in control conditions. Note that FK-506 inhibits P2X₃ current responses in KI neurons. D, Histograms of western blot signals obtained with anti-phosphorylated serine antibody applied to immunopurified P2X₃ receptors from WT and KI neurons in control condition and after incubation with FK-506 (5 μ M, 30 min). Note increase in serine phosphorylation after FK-506 treatment in KI neurons (n = 5, *p = 0.02). Data are normalized to the signals from WT neurons in control conditions. E, Histograms of western blot signals obtained with anti-phosphorylated threonine antibody show enhancement of threonine phosphorylation in WT (n = 5, *p = 0.046) as well as KI (n = 3, *p = 0.04) in neurons after treatment with FK-506. Data are normalized to the signals from WT neurons in control conditions.