Background
Methods
Cell Culturing and mutagenesis
Patch-clamp recordings in DRG neurons and transfected HEK293 cells
Voltage-clamp and macropatch recordings in Xenopus oocytes
Lipid binding assay
Biotinylation of surface proteins and Western blotting
Statistical analysis
Results
Depletion of PIP2 decreases P2X3 current density in DRG neurons
Control | Wortmannin | |
---|---|---|
DRG neurons
| 100.0 ± 9.3 | 20.4 ± 4.2 *** |
HEK293 cells
| 100.0 ± 9.2 | 107.6 ± 21.0 |
Xenopus oocytes
| 92.8 ± 6.3 | 94.2 ± 12.2 |
Intracellular PIP2 rescues native P2X3 current responses after wortmannin treatment
Depletion of phosphoinositides decreases P2X2/3 current density in DRG neurons
Phosphoinositides modulate P2X2/3 receptors in heterologous expression system
Control | 100 nM Wortmannin | 35 μM Wortmannin | |
---|---|---|---|
DRG neurons
| 100.0 ± 12.6 | 47.2 ± 7.6 ** | 40.9 ± 7.0 ** |
Xenopus oocytes
| 105.1 ± 5.5 | 66.3 ± 4.6 *** | 49.1 ± 3.2 *** |
PIP2 rescues P2X3 and P2X2 current responses from rundown in excised inside-out macropatches
No direct binding of phosphoinositides to the P2X3 subunit
Phosphoinositides modulate the rundown of P2X3 receptor currents in heterologous expression systems, which was reversed by mutation R356Q
Control | Wortmannin | |
---|---|---|
DRG neurons
| 96.0 ± 7.5 | 92.0 ± 5.3 |
HEK293 cells
| 89.9 ± 6.6 | 59.8 ± 5.4 ** |
Xenopus oocytes
| 93.1 ± 4.9 | 62.6 ± 7.0 ** |