Figure 4.

ω-agatoxin IVA restores anantin effects on P2X3 receptors in KI TG neurons. (a) Representative traces of α,β-meATP (10 µM, 2 s)-evoked P2X3 responses in control and after application of ω-agatoxin IVA (200 nM, 24 h) alone or in a combination with anantin (500 ng/mL; coapplied during the last 3 h of ω-agatoxin IVA treatment). Histograms on the right quantify current density values of P2X3 currents for each condition. Note that ω-agatoxin IVA reduces P2X3 currents and that anantin can up-regulate them to the control level (n = 23, 24, 25, *p < 0.05, Mann–Whitney rank sum test). (b) Western immunoblotting shows the amount of P2X3 pSer and total amount of P2X3 receptors in control, after 24 h ω-agatoxin IVA or ω-agatoxin IVA plus anantin (500 nM, 3 h) treatment. β-tubulin III was used as loading control. Histograms on the right show statistically higher P2X3 pSer value after ω-agatoxin IVA application compared to control (n = 4; *p < 0.05, Mann–Whitney rank sum test) with reversal by anantin application. (c) Top panel is a representative example of Western immunoblotting showing the amount of P2X3 receptors in raft (R) and non-raft (NR) membrane fractions in control and after treatment with ω-agatoxin IVA (200 nM, 24 h) or a combination of ω-agatoxin IVA and anantin (500 ng/mL; 3 h). Flotillin bands indicate lipid raft membrane fractions. Lower panel shows total amount of P2X3 receptors for each experimental condition; β-tubulin was used as loading control. Histograms on the right quantify mean P2X3 values in lipid raft and non-raft membrane fractions (n = 4; *p < 0.05, Kruskal–Wallis test).