Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels.

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating.

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

Sugar causes obesity and metabolic syndrome in mice independently of sweet taste.

Intake of sugars, especially the fructose component, is strongly associated with the development of obesity and metabolic syndrome, but the relative role of taste versus metabolism in driving preference, intake, and metabolic outcome is not fully understood. We aimed to evaluate the preference for sweet substances and the tendency to develop metabolic syndrome in response to these sugars in mice lacking functional taste signaling [P2X2 (P2X purinoreceptor 2)/P2X3 (P2X purinoreceptor 3) double knockout mice (DKO)] and mice unable to metabolize fructose (fructokinase knockout mice). Of interest, our data indicate that despite their inability to taste sweetness, P2X2/3 DKO mice still prefer caloric sugars (including fructose and glucose) to water in long-term testing, although with diminished preference compared with control mice. Despite reduced intake of caloric sugars by P2X2/3 DKO animals, the DKO mice still show increased levels of the sugar-dependent hormone FGF21 (fibroblast growth factor 21) in plasma and liver. Despite lower sugar intake, taste-blind mice develop severe features of metabolic syndrome due to reduced sensitivity to leptin, reduced ability to mobilize and oxidize fats, and increased hepatic de novo lipogenesis. In contrast to P2X2/3 DKO and wild-type mice, fructokinase knockout mice, which cannot metabolize fructose and are protected against fructose-induced metabolic syndrome, demonstrate reduced preference and intake for all fructose-containing sugars tested but not for glucose or artificial sweeteners. Based on these observations, we conclude that sugar can induce metabolic syndrome in mice independently of its sweet properties. Furthermore, our data demonstrate that the metabolism of fructose is necessary for sugar to drive intake and preference in mice.

Controlling Engineered P2X Receptors with Light.

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

Hearing loss mutations alter the functional properties of human P2X2 receptor channels through distinct mechanisms.

Activation of P2X2 receptor channels by extracellular ATP is thought to play important roles in cochlear adaptation to elevated sound levels and protection from overstimulation. Each subunit of a trimeric P2X2 receptor is composed of intracellular N and C termini, a large extracellular domain containing the ATP binding site and 2 transmembrane helices (TM1 and TM2) that form a cation permeable pore. Whole-exome sequencing and linkage analysis have identified 3 hP2X2 receptor mutations (V60L, D273Y, and G353R) that cause dominantly inherited progressive sensorineural hearing loss (DFNA41). Available structures of related P2X receptors suggest that these 3 mutations localize to TM1 (V60L), TM2 (G353R), or the ß-sheet linking the TMs to the extracellular ATP binding sites (D273Y). Previous studies have concluded that the V60L and G353R mutants are nonfunctional, whereas the D273Y mutant has yet to be studied. Here, we demonstrate that both V60L and G353R mutations do form functional channels, whereas the D273Y mutation prevents the expression of functional channels on the cell membrane. Our results show that the V60L mutant forms constitutively active channels that are insensitive to ATP or the antagonist suramin, suggesting uncoupling of the pore and the ligand binding domains. In contrast, the G353R mutant can be activated by ATP but exhibits alterations in sensitivity to ATP, inward rectification, and ion selectivity. Collectively, our results demonstrate that the loss of functional P2X2 receptors or distinct alterations of its functional properties lead to noise-induced hearing loss, highlighting the importance of these channels in preserving hearing.

Action of MK-7264 (gefapixant) at human P2X3 and P2X2/3 receptors and in vivo efficacy in models of sensitisation.

BACKGROUND AND PURPOSE: The P2X3 receptor is an ATP-gated ion channel expressed by sensory afferent neurons and is used as a target to treat chronic sensitisation conditions. The first-in-class, selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK-7264 (gefapixant), has progressed to Phase III trials for refractory or unexplained chronic cough. We used patch clamp to elucidate the pharmacology and kinetics of MK-7264 and rat models of hypersensitivity and hyperalgesia to test its efficacy on these conditions. EXPERIMENTAL APPROACH: Whole-cell patch clamp of 1321N1 cells expressing human P2X3 and P2X2/3 receptors was used to determine mode of MK-7264 action, potency, and kinetics. The analgesic efficacy was assessed using paw withdrawal threshold and limb weight distribution in rat models of inflammatory, osteoarthritic, and neuropathic sensitisation. KEY RESULTS: MK-7264 is a reversible allosteric antagonist at human P2X3 and P2X2/3 receptors. Experiments with the slowly desensitising P2X2/3 heteromer revealed concentration- and state-dependency to wash-on, with faster rates and greater inhibition when applied before agonist compared to during agonist application. The wash-on rate (τ value) for MK-7264 at maximal concentrations was much lower when applied before compared to during agonist application. In vivo, MK-7264 displayed efficacy comparable to naproxen in inflammatory and osteoarthritic sensitisation models and gabapentin in neuropathic sensitisation models, increasing paw withdrawal threshold and decreasing weight-bearing discomfort. CONCLUSIONS AND IMPLICATIONS: MK-7264 is a reversible and selective P2X3 and P2X2/3 antagonist, exerting allosteric antagonism via preferential activity at closed channels. Its efficacy in rat models supports its clinical investigation for chronic sensitisation conditions.

Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus.

Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-ß-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3ß2 (but not α4ß2) nAChR. Besides, P2X2 co-immunoprecipitated α3ß2 (but not α4ß2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3ß2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus.

Adenosine triphosphate drives head and neck cancer pain through P2X2/3 heterotrimers.

INTRODUCTION: Cancer pain creates a poor quality of life and decreases survival. The basic neurobiology of cancer pain is poorly understood. Adenosine triphosphate (ATP) and the ATP ionotropic receptor subunits, P2X2 and P2X3, mediate cancer pain in animal models; however, it is unknown whether this mechanism operates in human, and if so, what the relative contribution of P2X2- and P2X3-containing trimeric channels to cancer pain is. Here, we studied head and neck squamous cell carcinoma (HNSCC), which causes the highest level of function-induced pain relative to other types of cancer. RESULTS: We show that the human HNSCC tissues contain significantly increased levels of ATP compared to the matched normal tissues. The high levels of ATP are secreted by the cancer and positively correlate with self-reported function-induced pain in patients. The human HNSCC microenvironment is densely innervated by nerve fibers expressing both P2X2 and P2X3 subunits. In animal models of HNSCC we showed that ATP in the cancer microenvironment likely heightens pain perception through the P2X2/3 trimeric receptors. Nerve growth factor (NGF), another cancer-derived pain mediator found in both human and mouse HNSCC, induces P2X2 and P2X3 hypersensitivity and increases subunit expression in murine trigeminal ganglion (TG) neurons. CONCLUSIONS: These data identify a key peripheral mechanism in cancer pain and highlight the clinical potential of specifically targeting nociceptors expressing both P2X2 and P2X3 subunits (e.g., P2X2/3 heterotrimers) to alleviate cancer pain.

Direct gating of ATP-activated ion channels (P2X2 receptors) by lipophilic attachment at the outer end of the second transmembrane domain.

The ionic pore of the P2X receptor passes through the central axis of six transmembrane (TM) helices, two from each of three subunits. Val(48) and Ile(328) are at the outer end of TM1 and TM2, respectively. Homology models of the open and closed states of P2X2 indicate that pore opening is associated with a large lateral displacement of Ile(328). In addition, molecular dynamics simulations suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was activated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-d-glucamine. The ATP-binding residue Lys(69) was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) receptors. The propyl-MTS did not open P2X2 receptors in which the Val(48) side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val(48) and Ile(328) stabilizes the closed channel and that this is broken by covalent attachment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during channel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the channel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the position of the TMs within the bilayer.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Astrocyte-derived ATP modulates depressive-like behaviors.

Major depressive disorder (MDD) is a cause of disability that affects approximately 16% of the world's population; however, little is known regarding the underlying biology of this disorder. Animal studies, postmortem brain analyses and imaging studies of patients with depression have implicated glial dysfunction in MDD pathophysiology. However, the molecular mechanisms through which astrocytes modulate depressive behaviors are largely uncharacterized. Here, we identified ATP as a key factor involved in astrocytic modulation of depressive-like behavior in adult mice. We observed low ATP abundance in the brains of mice that were susceptible to chronic social defeat. Furthermore, we found that the administration of ATP induced a rapid antidepressant-like effect in these mice. Both a lack of inositol 1,4,5-trisphosphate receptor type 2 and transgenic blockage of vesicular gliotransmission induced deficiencies in astrocytic ATP release, causing depressive-like behaviors that could be rescued via the administration of ATP. Using transgenic mice that express a Gq G protein-coupled receptor only in astrocytes to enable selective activation of astrocytic Ca(2+) signaling, we found that stimulating endogenous ATP release from astrocytes induced antidepressant-like effects in mouse models of depression. Moreover, we found that P2X2 receptors in the medial prefrontal cortex mediated the antidepressant-like effects of ATP. These results highlight astrocytic ATP release as a biological mechanism of MDD.

Mutation of the ATP-gated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise.

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X(2) receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X(2) receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X(2) function is required for life-long normal hearing and for protection from exposure to noise.

Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closed-to-open transition of the P2X2 receptor.

P2X receptors are trimeric adenosine-5'-triphosphate (ATP)-gated cation channels involved in fast signal transduction in many cell types. In this study, we used homology modeling of the rat P2X2 receptor with the zebrafish P2X4 X-ray template to determine that the side chains of the Glu167 and Arg290 residues are in close spatial vicinity within the ATP-binding pocket when the rat P2X2 channel is closed. Through charge reversal mutation analysis and mutant cycle analysis, we obtained evidence that Glu167 and Arg290 form an electrostatic interaction. In addition, disulfide trapping indicated the close proximity of Glu167 and Arg290 when the channel is in the closed state, but not in the ATP-bound open state. Consistent with a gating-induced movement that disrupts the Glu167/Arg290 salt bridge, a comparison of the closed and open rat P2X2 receptor models revealed a significant rearrangement of the protein backbone and the side chains of the Glu167 and Arg290 residues during the closed-to-open transition. The associated release of the Glu167/Arg290 salt bridge during channel opening allows a strong ionic interaction between Arg290 and a γ-phosphate oxygen of ATP. We conclude from these results that the state-dependent salt bridge switching from Arg290/Glu167 to Arg290/ATP fulfills a dual role: to destabilize the closed state of the receptor and to promote the ionic coordination of ATP in the ATP-binding pocket.

P2X1 and P2X2 receptors in the central nervous system as possible drug targets.

P2X receptors are homo- or heterotrimeric ATP-gated cation channels that assemble from seven subunits, P2X1-P2X7. To our knowledge, no drug that acts on the P2X1 or P2X2 receptors in the CNS or elsewhere in the body has been approved, nor is there such a drug currently in clinical trials. Only a few non-drug-like antagonists such as the suramin derivatives NF449 and NF770 and the anthraquinone derivative PSB-1011 are available as pharmacological tools to block the P2X1 and P2X2 receptors, respectively. The focus of this review is twofold. First, we review the current knowledge of the role of the P2X1 and P2X2 receptors in normal and pathological CNS functions as inferred from experiments with wild-type, P2X1 knockout and P2X2 knockout mice. From the available data we conclude that the P2X1 and P2X2 receptors may have therapeutic potential as targets for neuroprotective drugs. Second, we review the impact of the recent resolution of the crystal structure of the zebrafish P2X4 receptor in the apo closed state and the ATP-bound open state. The P2X4 crystal structure opens the exciting possibility to generate P2X homology models for a rational drug design. In silico docking experiments with a homology-modeled rat P2X2 receptor revealed an almost perfect coordination of the nanomolar potent P2X2 antagonist NF770 through strong polar interactions between the acidic groups of NF770 and the mostly basic groups of the ATP-binding pocket. Such structural information might be helpful in designing drug-like compounds that function as selective P2X receptor antagonists without the pharmacokinetic limitations of the currently available antagonists.

Allosteric nature of P2X receptor activation probed by photoaffinity labelling.

BACKGROUND AND PURPOSE: In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate. EXPERIMENTAL APPROACH: We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors. KEY RESULTS: Irradiation of the P2X2/1 receptor chimera - BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists. CONCLUSIONS AND IMPLICATIONS: Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding.

Modulation of bladder afferent signals in normal and spinal cord-injured rats by purinergic P2X3 and P2X2/3 receptors.

OBJECTIVE: To evaluate the role of bladder sensory purinergic P2X3 and P2X2/3 receptors on modulating the activity of lumbosacral neurones and urinary bladder contractions in vivo in normal or spinal cord-injured (SCI) rats with neurogenic bladder overactivity. MATERIALS AND METHODS: SCI was induced in female rats by complete transection at T8-T9 and experiments were performed 4 weeks later, when bladder overactivity developed. Non-transected rats were used as controls (normal rats). Neural activity was recorded in the dorsal horn of the spinal cord and field potentials were acquired in response to intravesical pressure steps via a suprapubic catheter. Field potentials were recorded under control conditions, after stimulation of bladder mucosal purinergic receptors with intravesical ATP (1 mm), and after intravenous injection of the P2X3/P2X2/3 antagonist AF-353 (10 mg/kg and 20 mg/kg). Cystometry was performed in urethane-anaesthetised rats intravesically infused with saline. AF-353 (10 mg/kg) was systemically applied after baseline recordings; the rats also received a second dose of AF-353 (20 mg/kg). Changes in the frequency of voiding (VC) and non-voiding (NVC) contractions were evaluated. RESULTS: SCI rats had significantly higher frequencies for field potentials and NVC than NL rats. Intravesical ATP increased field potential frequency in control but not SCI rats, while systemic AF-353 significantly reduced this parameter in both groups. AF-353 also reduced the inter-contractile interval in control but not in SCI rats; however, the frequency of NVC in SCI rats was significantly reduced. CONCLUSION: The P2X3/P2X2/3 receptors on bladder afferent nerves positively regulate sensory activity and NVCs in overactive bladders.

Gating properties of the P2X2a and P2X2b receptor channels: experiments and mathematical modeling.

Adenosine triphosphate (ATP)-gated P2X2 receptors exhibit two opposite activation-dependent changes, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of calcium and the C-terminal domain in gating, we combined biophysical and mathematical approaches using two splice forms of receptors: the full-size form (P2X2aR) and the shorter form missing 69 residues in the C-terminal domain (P2X2bR). Both receptors developed conductivity for N-methyl-D-glucamine within 2-6 s of ATP application. However, pore dilation was accompanied with a decrease rather than an increase in the total conductance, which temporally coincided with rapid and partial desensitization. During sustained agonist application, receptors continued to desensitize in calcium-independent and calcium-dependent modes. Calcium-independent desensitization was more pronounced in P2X2bR, and calcium-dependent desensitization was more pronounced in P2X2aR. In whole cell recording, we also observed use-dependent facilitation of desensitization of both receptors. Such behavior was accounted for by a 16-state Markov kinetic model describing ATP binding/unbinding and activation/desensitization. The model assumes that naive receptors open when two to three ATP molecules bind and undergo calcium-independent desensitization, causing a decrease in the total conductance, or pore dilation, causing a shift in the reversal potential. In calcium-containing media, receptor desensitization is facilitated and the use-dependent desensitization can be modeled by a calcium-dependent toggle switch. The experiments and the model together provide a rationale for the lack of sustained current growth in dilating P2X2Rs and show that receptors in the dilated state can also desensitize in the presence of calcium.

Tightening of the ATP-binding sites induces the opening of P2X receptor channels.

The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

A possible role of the cholinergic and purinergic receptor interaction in the regulation of the rat urinary bladder function.

The contractile activation of the upper (dome) and lower (base) parts of the urinary bladder show some differences. Cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the rat urinary bladder were investigated. Smooth muscle cells were thus freshly isolated or cultured both from the dome and the base of the rat urinary bladder and the contribution from cholinergic and purinergic pathways to their Ca(2+) homeostasis was examined. The expression of nicotinic acetylcholine (nAChR) and P2X2 purinergic receptors on the cultured cells and on tissue sections was investigated. The ATP-evoked Ca(2+) transients in rat smooth muscle cells did not show any desensitization. However, when ATP was administered together with carbamylcholine (CCh), the latter essentially prevented ATP from evoking Ca(2+) transients in smooth muscle cells from the base (suppression to 12 ± 2.5% of control, n = 57; p < 0.01), but not from the dome (99 ± 5% of control, n = 52; p > 0.05) of the rat urinary bladder. While atropine was unable to modify (6 ± 3% of control, n = 14; p < 0.05), α-bungarotoxin (118 ± 12% of control, n = 20; p > 0.05) blocked the inhibitory effects of CCh. Additionally, α7 subunits of nAChR and P2X2 purinergic receptors were identified using immunocytochemistry, immunohistochemistry, and Western blot in cultured urinary bladder smooth muscle cells, in urinary bladder sections, and in urinary bladder muscle strips, respectively, suggesting that the activation of nAChR modifies the action of ATP.

P2X purinergic receptor knockout mice reveal endogenous ATP modulation of both vasopressin and oxytocin release from the intact neurohypophysis.

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca(2+) currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback-mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically-stimulated neuropeptide release. Intact neurohypophyses (NH) from wild-type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25-s bursts (3 V at 39 Hz) separated by 21-s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically-stimulated AVP release. A similar inhibition by suramin was observed in electrically-stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically-stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically-stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this 'selective' antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive-feedback mechanism to 'differentially' modulate neuropeptide release from central nervous system terminals.