Claudio Coddou

Activation and Regulation of Purinergic P2X Receptor Channels

Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.

Small molecule positive allosteric modulation of TRPV1 activation by vanilloids and acidic pH

Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system and functions as a multimodal nociceptor gated by temperatures greater than 43°C, protons, and small-molecule vanilloid ligands such as capsaicin. The ability to respond to heat, low pH, vanilloids, and endovanilloids and altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics and resulted in the discovery of potent antagonists. In human clinical trials, observations of hyperthermia and the potential for thermal damage by suppressing the ability to sense noxious heat suggested that full-scale blockade of TRPV1 function can be counterproductive and subtler pharmacological approaches are necessary. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory-mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by capsazepine or ruthenium red and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation.

Reactive Oxygen Species Potentiate the P2X2 Receptor Activity through Intracellular Cys430

P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69 residue segment in the C terminal, termed P2X2b, as the donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera but was absent in oocytes expressing P2X4/2b chimera. Site-directed mutagenesis experiments revealed that the Cys430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H2O2) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents but not P2X2bR and P2X2a–C430A and P2X2a–C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H2O2 potentiation. Alkylation of Cys430 residue with methylmethane-thiosulfonate also abolished the mercury and H2O2 potentiation. Altogether, these results are consistent with the hypothesis that the Cys430 residue is an intracellular P2X2aR redox sensor.

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.

Role of domain calcium in purinergic P2X2 receptor channel desensitization

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na(+) with N-methyl-d-glucamine (NMDG(+)), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from -60 to +120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.

Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.

Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP

Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell‐specific and tonotopically‐determined manner. Developmental down‐regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high‐to‐low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP‐AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors.

Cyclin - dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C - terminal threonine 372

Abstract The purinergic P2X2 receptor (P2X2R) is an adenosine triphosphate–gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (372TPKH375), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca2+ in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 μM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 μM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 μM concentration but not 0.75 μM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.