A Mechanism of Intracellular P2X Receptor Activation

P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen.     

Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells

The P2X4 receptor (P2X4R) is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane (TM) domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. P2X4Rs activate rapidly and desensitize within the seconds of agonist application, both with the rates dependent on ATP concentrations, and deactivate rapidly and independently of ATP concentration. Disruption of conserved cysteine ectodomain residues affects ATP binding and gating. Several ectodomain residues of P2X4R were identified as critical for ATP binding, including K67, K313, and R295. Ectodomain residues also account for the allosteric regulation of P2X4R; H140 is responsible for copper binding and H286 regulates receptor functions with protons. Ivermectin sensitized receptors, amplified the current amplitude, and slowed receptor deactivation by binding in the TM region. Scanning mutagenesis of TMs revealed the helical topology of both domains, and suggested that receptor function is critically dependent on the conserved Y42 residue. In this brief article, we summarize this study and re-interpret it using a model based on crystallization of the zebrafish P2X4.1 receptor.     

Imaging P2X4 receptor subcellular distribution,trafficking, and regulation using P2X4-pHluorin

P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.     

P2X receptors mediated abnormal interaction between satellite glial cells and neurons in visceral pathological changes

The adenosine triphosphate (ATP)‐gated P2X receptor cation channel family consists of permeable ligand‐gated ion channels that expand on the binding of extracellular adenosine 5’‐ATP. ATP‐gated P2X receptors are trimer ion channels that assemble homo or isomer from seven cloned subunits. P2X receptors are discovered mostly in mammalian and are being found in an increasing number of non‐vertebrates, such as zebrafish, bullfrog, and ameba. P2X receptors are involved in many physiological processes, including regulation of heart rhythm and contractility, and regulation of pain, especially chronic pain and glia integration. This review summarizes the current studies on the regulation of P2X receptors in abnormal neuronal‐glial interaction and the pathological changes in viscera, especially in myocardial ischemia.     

CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.     

Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits.

The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocytochemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X1 and P2X5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X1 and P2X5 subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X1 and the kinetics of P2X5. When expressed in Xenopus oocytes, hetero-oligomeric P2X1+5 ATP receptors were characterized by slowly desensitizing currents highly sensitive to the agonist alpha,beta-methylene ATP (EC50 = 1.1 microM) and to the antagonist trinitrophenyl ATP (IC50 = 64 nM), observed with neither P2X1 nor P2X5 alone. Direct physical evidence for P2X1+5 co-assembly was provided by reciprocal subunit-specific co-purifications between epitope-tagged P2X1 and P2X5 subunits transfected in HEK-293A cells.     

Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids

Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green–dextran–tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.     

Imaging P2X4 receptor lateral mobility in microglia: regulation by calcium and p38 MAPK

ATP-gated ionotropic P2X4 receptors are up-regulated in activated microglia and are critical for the development of neuropathic pain, a microglia-associated disorder. However, the nature of how plasma membrane P2X4 receptors are regulated in microglia is not fully understood. We used single-molecule imaging to track quantum dot-labeled P2X4 receptors to explore P2X4 receptor mobility in the processes of resting and activated microglia. We find that plasma membrane P2X4 receptor lateral mobility in resting microglial processes is largely random, consisting of mobile and slowly mobile receptors. Moreover, lateral mobility is P2X subunit- and cell-specific, increased in an ATP activation and calcium-dependent manner, and enhanced in activated microglia by the p38 MAPK pathway that selectively regulates slowly mobile receptors. Thus, our data indicate that P2X4 receptors are dynamically regulated mobile ATP sensors, sampling more of the plasma membrane in response to ATP and during the activated state of microglia that is associated with nervous system dysfunction.     

Cytolytic P2X purinoceptors

Anecdoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloride-sensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.     

Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes.

Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate-dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H(+)-ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9-6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non-proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.     

Surface-targeted lysosomal membrane glycoprotein-1 (Lamp-1) enhances lysosome exocytosis and cell invasion by Trypanosoma cruzi

To gain entry into non-phagocytic cells, Trypanosoma cruzi trypomastigotes recruit lysosomes to the host cell surface. Lysosome fusion at the site of parasite entry leads to the formation of a parasitophorous vacuole with lysosomal properties. Here, we show that increased expression of the lysosomal membrane glycoprotein Lamp-1 at the cell surface renders CHO cells more susceptible to trypomastigote invasion in a microtubule-dependent fashion. Mutation of critical residues in the lysosome-targeting motif of Lamp-1 abolished the enhancement of T. cruzi invasion. This suggests that interactions dependent on Lamp-1 cytoplasmic tail motifs, and not the surface-exposed luminal domain, modulate T. cruzi entry. Measurements of Ca2+-triggered exocytosis of lysosomes in these cell lines revealed an enhancement of beta-hexosaminidase release in cells expressing wild-type Lamp-1 on the plasma membrane; this effect was not observed in cell lines transfected with Lamp-1 cytoplasmic tail mutants. These results also implicate Ca2+-regulated lysosome exocytosis in cell invasion by T. cruzi and indicate a role for the Lamp-1 cytosolic domain in promoting more efficient fusion of lysosomes with the plasma membrane.     

Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

Covalent Modification of Mutant Rat P2X2 Receptors with a Thiol-Reactive Fluorophore Allows Channel Activation by Zinc or Acidic pH without ATP

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C₅-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

A protein kinase C site highly conserved in P2X subunits controls the desensitization kinetics of P2X(2) ATP-gated channels

P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording in Xenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X(2) receptors. Mutant receptors P2X(2)T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X(2) receptors with truncated C terminus exhibited variable cell-specific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr(18) was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X(2) subunits are necessary for the full expression of slowly desensitizing ATP-gated channels.     

Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.     

Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex

The purinergic receptor P2X(7) is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X(7) receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X(7)R in Xenopus oocytes results exclusively in formation of a non-selective cation channel. However, here we show that co-expression of P2X(7)R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X(7)R co-expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X(7)R signaling complex.     

Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)

Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y(2) receptors (Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger J. J Am Soc Nephrol 18: 2062-2070, 2007). In addition, we found evidence for a basolateral P2X receptor. Here, we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short-circuit current (I'(sc)). Nonstimulated mTALs transported at a rate of 1,197 ± 104 μA/cm(2) (n = 10), which was completely blockable with luminal furosemide (100 μM). Basolateral ATP (100 μM) acutely (1 min) and reversibly reduced the absorptive I'(sc). After 2 min, the reduction amounted to 24.4 ± 4.0% (n = 10). The nonselective P2 receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2-methylthio ATP mimicked the ATP effect, and the P2X receptor antagonist periodate-oxidized ATP blocked it. In P2X(7)(-/-) mice, the ATP effect remained unaltered. In contrast, in P2X(4)(-/-) mice the ATP-induced inhibition of transport was reduced. A comprehensive molecular search identified P2X(4), P2X(5), and P2X(1) receptor subunit mRNA in isolated mouse mTALs. These data define that basolateral ATP exerts a significant inhibition of Na(+) absorption in mouse mTAL. Pharmacological, molecular, and knockout mouse data identify a role for the P2X(4) receptor. We suggest that other P2X subunits like P2X(5) are part of the P2X receptor complex. These data provide the novel perspective that an ionotropic receptor and thus a nonselective cation channel causes transport inhibition in an intact renal epithelium.