Subtype-Specific Regulation of P2X3 and P2X2/3 Receptors by Phosphoinositides in Peripheral Nociceptors

Abstract

BackgroundP2X3 and P2X2/3 purinergic receptor-channels, expressed in primary sensory neurons that mediate nociception, have been implicated in neuropathic and inflammatory pain responses. The phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) are involved in functional modulation of several types of ion channels. We report here evidence that these phospholipids are able to modulate the function of homomeric P2X3 and heteromeric P2X2/3 purinoceptors expressed in dorsal root ganglion (DRG) nociceptors and in heterologous expression systems.ResultsIn dissociated rat DRG neurons, incubation with the PI3K/PI4K inhibitor wortmannin at 35 μM induced a dramatic decrease in the amplitude of ATP- or α,β-meATP-evoked P2X3 currents, while incubation with 100 nM wortmannin (selective PI3K inhibition) produced no significant effect. Intracellular application of PIP2 was able to fully reverse the inhibition of P2X3 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 μM wortmannin incubation induced a significant decrease in the rate of receptor recovery. Native and recombinant P2X2/3 receptor-mediated currents were inhibited by incubation with wortmannin both at 35 μM and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partially reversed by application of PIP2 or PIP3, indicating a sensitivity to both phosphoinositides in DRG neurons and Xenopus oocytes. Using a lipid binding assay, we demonstrate that the C-terminus of the P2X2 subunit binds directly to PIP2, PIP3 and other phosphoinositides. In contrast, no direct binding was detected between the C-terminus of P2X3 subunit and phosphoinositides.ConclusionOur findings indicate a functional regulation of homomeric P2X3 and heteromeric P2X2/3 ATP receptors by phosphoinositides in the plasma membrane of DRG nociceptors, based on subtype-specific mechanisms of direct and indirect lipid sensing.

Activation and Regulation of Purinergic P2X Receptor Channels

“…Likewise, P2X3R and P2X7R are inhibited by decreases in PIP 2 induced either by PI4K inhibition or phospholipase C activation by coexpressed G protein coupled receptors (Zhao et al, 2007a,b). Native and recombinant P2X2/3Rs are also regulated by phosphoinositides (Mo et al, 2009).…”

Co-localization of Pirt protein and P2X2 receptors in the mouse enteric nervous system

“…Previous studies have implied that phosphoinositides may be one of the intermediaries between the two proteins. For instance, it has been found that the C-terminus of the P2X2 receptor binds directly to phosphatidyl-inositol-4,5-bisphosphate (PIP2), PIP3, and other phosphoinositides [21]. By expressing the target channel in Xenopus oocytes, P2X2 receptors were shown to be activated by PIP2 [22], and other work has revealed that heterotrimeric P2X2/3 receptors are also regulated by phosphoinositides [21].…”

“…For instance, it has been found that the C-terminus of the P2X2 receptor binds directly to phosphatidyl-inositol-4,5-bisphosphate (PIP2), PIP3, and other phosphoinositides [21]. By expressing the target channel in Xenopus oocytes, P2X2 receptors were shown to be activated by PIP2 [22], and other work has revealed that heterotrimeric P2X2/3 receptors are also regulated by phosphoinositides [21]. It has also been shown that the C terminus of Pirt protein binds to several phosphoinositides, including PIP2 [15,23,22,24,21,25] This modulation of P2X receptors and Pirt protein by phospholipids may explain their roles under conditions such as chronic pain and immune disorders.…”

Molecular mechanism for opioid dichotomy: bidirectional effect of μ-opioid receptors on P2X3 receptor currents in rat sensory neurones

“…Depletion of the membrane pool of PIP 2 leads to the inhibition of the P2X 3 current, which can be restored by addition of PIP 2 into the cytosol [37]; activation of MOR leads to PLC- Exposure of PTX-treated cells to Enk increased the peak amplitude of P2X 3 currents, which action was antagonized by staurosporine and U73122. *P<0.05, statistically significant difference (n=5) mediated hydrolyses of PIP 2 thus depleting the membrane pool of the latter.…”

“…It was proposed that molecular components of G protein/PLC signalling pathways and their final targets can be segregated between spatially separated microdomains [42], while distinct pools of PIP 2 in plasma membrane have been identified by electron microscopy [43]. Depletion of the PIP 2 pool associated with P2X 3 receptors leads to the inhibition of P2X 3 current [37]. We suggest that hydrolysis of another PIP 2 indicating intracellular release) was attenuated to a much lesser degree pool separated from P2X 3 receptors induces their stimulation via the DAG/PKC pathway (Fig.…”