Phosphorylation state of P2X
receptors in KI neurons. A, Example of western blots of P2X3 receptors immunopurified from WT and KI neurons and probed with anti-phosphorylated serine antibodies. Note decreased serine phosphorylation of KI samples, a phenomenon prevented by pre-treatment with ω-agatoxin (200 nM, 24 h). B, Histograms quantify serine phosphorylation state of P2X3 receptors (*p = 0.02 vs. WT; n = 5) that is enhanced for KI receptors and prevented by ω-agatoxin (n = 3, #p = 0.027). C, Example of western blots of immunopurified P2X3 receptors from WT and KI neurons probed with anti-phosphorylated threonine (top row) or tyrosine (middle row) antibodies. Total P2X3 receptor levels are shown in bottom row. Control antibody lane (Ab) is also shown. n = 8 for threonine and n = 3 for tyrosine. D, Pretreatment of neurons with the CaMKII inhibitor KN-93 (5 μ M, 90 min) restores the P2X3 serine phosphorylation state without changing total P2X3 receptor expression. Histograms (right) quantify serine phosphorylation state of P2X3 receptors, an effect blocked by KN-93 (n = 3; *p = 0.02; #p = 0.017) in KI neurons. E, Representative examples of WT and KI current traces evoked by α,β-meATP (10 μ M, 2-s application) in control (Ctr) and after pre-treatment with KN-93 (5 μ M, 90 min). Histograms (right) show the percent inhibition by KN-93 of peak amplitude of α,β-meATP-mediated P2X3 receptor currents in KI neurons (n = 22), that is significantly larger in KI cells (n = 17, *p = 0.039).