A dual polybasic motif determines phosphoinositide binding and regulation in the P2X channel family.

Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids.

Modulation of heteromeric P2X1/5 receptors by phosphoinositides in astrocytes depends on the P2X1 subunit.

Purinergic signaling is critical for neuron-glia communication. Glial cells participate in synaptic transmission and express metabotropic P2Y as well as ionotropic P2X ATP receptors. In astrocytes, endogenous ATP-evoked currents with kinetics and pharmacology characteristic of the heteromeric P2X1/5 receptor channel have recently been reported. We investigated the interaction of major phosphoinositides with heteromeric P2X1/5 channels. Using patch-clamp electrophysiology on enhanced green fluorescent protein-expressing astrocytes acutely isolated from cortical slices of transgenic mice, we report a strong modulation of P2X1/5-like currents by phosphoinositides. Wortmannin-induced depletion of phosphoinositides decreases the amplitude of both the fast and sustained component of the P2X1/5-like currents although recovery and kinetics remain intact. In transfected human embryonic kidney cells, we provide evidence that depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] levels significantly decreases P2X1/5 currents while intracellular application of PI(4,5)P(2) completely rescued P2X1/5 currents, ruling out the involvement of phosphatidylinositol 3,4,5-trisphosphate. In contrast to P2X1, homomeric P2X5 current responses were found insensitive to phosphoinositides, and the C-terminus of P2X5 subunit lacked binding to phospholipids in an overlay assay. Our results suggest that the contribution of calcium-permeable heteromeric P2X1/5 receptor channels to the excitability of astrocytes is modulated by PI(4,5)P(2) through the P2X1 lipid-binding domain.

Phosphoinositides regulate P2X4 ATP-gated channels through direct interactions.

P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X(4) subtype, upregulated in activated microglia, is thought to play a critical role in the development of tactile allodynia following peripheral nerve injury. Posttranslational regulation of P2X(4) function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P(2) (PIP(2)) and PI(3,4,5)P(3) (PIP(3)), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X(4) receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP(2) and PIP(3) with wortmannin significantly decreased P2X(4) current amplitude and P2X(4)-mediated calcium entry measured in patch clamp recordings and ratiometric ion imaging, respectively. Wortmannin-induced depletion of phosphoinositides in Xenopus oocytes decreased the current amplitude of P2X(4) responses by converting ATP into a partial agonist. It also decreased their recovery from desensitization and affected their kinetics. Injection of phosphoinositides in wortmannin-treated oocytes reversed these effects and application of PIP(2) on excised inside-out macropatches rescued P2X(4) currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X(4) subunit (Cys(360)-Val(375)) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP(2) and PIP(3) on P2X(4) function through direct channel-lipid interactions.

Direct modulation of P2X1 receptor-channels by the lipid phosphatidylinositol 4,5-bisphosphate.

The P2X(1) receptor-channels activated by extracellular ATP contribute to the neurogenic component of smooth muscle contraction in vascular beds and genitourinary tracts of rodents and humans. In the present study, we investigated the interactions of plasma membrane phosphoinositides with P2X(1) ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X(1)-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P(2) levels with wortmannin significantly decreases P2X(1) current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X(1) currents by phospholipase C-coupled 5-hydroxytryptamine(2A) metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X(1) activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X(1) currents could be reversed by addition of intracellular PI(4,5)P(2), but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P(2) to excised inside-out macropatches rescued P2X(1) currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X(1) subunit directly binds to PI(4,5)P(2) and other anionic phospholipids, and we identified the basic residue Lys(364) as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P(2) plays a key role in the expression of full native and heterologous P2X(1) function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.

Differential regulation of microglial P2X4 and P2X7 ATP receptors following LPS-induced activation.

Activation of microglia has been implicated in many neurological conditions including Alzheimer's disease and neuropathic pain. Recent studies provide evidence that P2X ATP receptors on the surface of microglia play a crucial role in initiation of inflammatory cascades. We investigated changes in surface P2X receptors in BV-2 murine microglial cells following their activation by pro-inflammatory bacterial lipopolysaccharides (LPS). mRNA analysis using RT-PCR confirmed the presence of P2X4 and P2X7 as the main P2X subunits. Application of ATP at low (< or =100 microM) and high (> or =1 mM) concentrations, as well as BzATP, activated inward currents in BV-2 cells. Current responses of P2X4 and P2X7 subtypes could be distinguished based on their respective sensitivity to the positive modulator ivermectin and to the antagonist Brilliant Blue G. Treatment of BV-2 cells with LPS leads to a transient increase in ivermectin-sensitive P2X4 currents, while dominant P2X7 currents remain largely unaffected. This increase in P2X4 function was concomitant with higher receptor protein expression, itself related to an upregulation of P2X4 mRNA levels that peaked at 48 h post-LPS treatment. Our data demonstrate that although LPS activation has a minor impact on P2X7 receptors that remain the major ionotropic ATP receptors in microglia, it specifically enhances responses to low ATP concentrations mediated by P2X4 receptors, highlighting the significant contribution of both subtypes to neuroinflammatory mechanisms and pathologies.

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons.

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.

High zinc sensitivity and pore formation in an invertebrate P2X receptor.

To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn2+ ions (IC50 = 0.4 microM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.

Selective knock-down of P2X7 ATP receptor function by dominant-negative subunits.

Among the family of P2X ATP-gated cation channels, the P2X7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type characteristics in both agonist affinity and ionic selectivity, strongly suggesting that the heteromeric channels are functionally impaired. The mutant subunits also suppressed the P2X7-dependent pore formation as assessed by uptake of the propidium dye YO-PRO-1 (Molecular Probes, Eugene, OR) in response to 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) in transfected human embryonic kidney 293 cells. Native responses to BzATP as well as ATP-induced ethidium dye uptake were significantly knocked down (31 +/- 9% and 25 +/- 7% of control, respectively) in mouse macrophage cell line RAW264.7 transfected with the mutant subunits. Therefore, these dominant-negative subunits provide selective genetic tools to investigate the functional roles of native P2X7 receptors.

Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels.

Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the functional interaction between native P2X2 and 5-HT3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.