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Highlights in basic autonomic neurosciences: nucleotide receptors: molecular signalling and structural elements
Autonomic Neuroscience: Basic and Clinical 158 (2010) 1–4
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
HIGHLIGHTS IN BASIC AUTONOMIC NEUROSCIENCES: NUCLEOTIDE RECEPTORS: MOLECULAR SIGNALLING AND STRUCTURAL ELEMENTS Prepared by: J. Pablo Huidobro-Toro Nucleotide Research Labs, Department of Physiology, Faculty of Biological Sciences, P. Catholic University of Chile, Casilla 114-D, Santiago, Chile Section Editor: Michael P. Gilbey
Extracellular nucleotides such as ATP, UTP, ADP and UDP have attracted much attention as novel messengers of non-excitable and excitable cells, including peripheral autonomic and central nerve endings. Nucleotides are released into the extracellular milieu by a variety of mechanisms involving channels, transporters and synaptic vesicles; these molecules act as auto/paracrine signals. A set of at least 15 plasma membrane receptors respond to extracellular nucleotides and belong to either the ATP-gated ionic channels, which form either homo- or heteromeric receptors (P2XR1–7 subtypes), or are G-protein coupled (eight clones of P2YR receptor subtypes), most of these receptors oligomerize. These receptors do not require γ phosphate hydrolysis to signal intracellularly and are involved in many autonomic control functions including those of blood flow, bladder voiding, intestinal motility and secretion, and airway conductance.
Kawate T., Michel J.C., Birdsong, W., Gouaux, E. (2009). Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460: 592–598. Article summary P2X receptors are a novel family of cation-selective ion channels gated by extracellular ATP. Because P2X receptors are not related to other ion channel proteins of known structure, it was a challenge to crystallize a representative family member and examine the crystals by X rays to decipher its 3-D structure. To improve crystallization, the authors took advantage of zebrafish P2X4R deletion mutants lacking both the N and C terminus intracellular domains; mutants maintained unaltered the complete extracellular and transmembrane domains. The chalice-shaped trimeric receptor is composed of three entangled subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in beta-strands, have large acidic patches that may attract cations through fenestrations to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an approximately 8 Å slab of protein. The location of three non-canonical, intersubunit ATP-binding sites was defined, suggesting that ATP binding promotes subunit rearrangement and ion 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.08.001
channel opening. The orthosteric ATP binding site is not in the immediate vicinity of the receptor transmembrane domain. Commentary The crystal structure of this ATP-gated P2X ion channel was analyzed in its close, resting state, with a 3.1 Å resolution; it provided unequivocal evidence, at an atomic scale, of the trimetric subunit channel stoichiometry, confirming prior empirical biochemical analysis. The authors compare the 3-D receptor contour of a single subunit to a dolphin-like shape. They reveal with precision, the 3-D organization of the large extracellular domain of each subunit and the interfaces between adjacent subunits. The putative orthosteric site for ATP binding was tentatively localized in the outer part of the extracellular domain; likely nucleotide binding induces conformational changes within and between subunits such that the pore topology accommodates for cation influx. The exact spatial location of the 12 highly conserved extracellular cysteines was revealed and which of these residues form each of the six predicted S–S bond pairs of the extracellular domain was determined. The level of resolution permitted the visualization of the 3-D intersubunit contacts required to form the channel pore independent of homo- or heteromer subunit assembly. The 3-D viewing allowed an insight into the pore entry identifying an upper, central and an extracellular vestibule in the extracellular subunit domain linked to the intracellular vestibule explaining how the ions permeate the cell. The channel pore is located on the molecular three-fold axis, with the extracellular vestibule connected to the bulk solution through a fenestration, allowing the entrance of cations. The proposed P2X channel receptor model allowed the further use of other bioinformatics programs to further investigate and identify through modelling the putative spatial localization of trace metal allosteric modulator sites which had been proposed to either increase or decrease the receptor affinity for ATP. Surprisingly when the reported amino acids suspected to conform part of the trace metal coordination site were identified and visualized in the P2X4R 3-D structure, they were found to be located in the immediate vicinity of the purported ATP binding site, in agreement with Coddou et al. (J. Biol. Chem., 282: 36879–36886, 2007). However, and far more importantly, the structural backbone of the P2X4R will allow the modelling of the complete P2X receptors, a necessary first
2
J.P. Huidobro-Toro / Autonomic Neuroscience: Basic and Clinical 158 (2010) 1–4
step towards the goal of rational drug design, and will provide essential data required to understand the details of pore opening kinetics and explain the electrophysiological properties of each channel subtypes and their fine intricacies at the molecular level. This paper is a classic in the field due to its three-dimensional visualization of the P2X4R conformation.
Choi R.C.Y., Simon J., Tsim K.W.K. Barnard E.A. (2008). Constitutive and agonist-induced dimerization of the P2Y1 receptor: relationship to internalization and scaffolding. J. Biol. Chem., 283: 11050–11063. Article summary Although initially visualized as independent cell membrane proteins, G-protein coupled receptors are now generally thought as dimers forming either homo- or heterooligomers. This study was the first to address the formation of P2Y receptor oligomers and assessed whether selective receptor activation with preferential ligands modifies the state of receptor dimerization. In living cells, P2Y1R dimerization was quantified by an improved version of fluorescence resonance energy transfer donor photobleaching examination. In the resting state, 44% of the P2Y1Rs expressed in HEK293 cell membranes are dimers; however, agonist exposure induced up to 85–100% dimerization. Monomer and constitutive dimers are fully active in cell signalling. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonists, an indication of a reversible process. Receptor dimers are required for internalization of the P2Y1R at 37 °C; at 20 °C dimerization also occurs, but endocytosis is abolished. Removal of the Cterminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. Distinction should therefore be made between 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signalling membrane micro-domain, and 2) free dimers in the cell membrane, which may dimerize by agonist exposure. In summary, for class A G-protein coupled receptors, the authors suggest that the percentages of free monomers, although variable, may oscillate up to 40%; agonist induced dimers are variable and may amount to as much as 100% of the cell membrane receptor population.
receptors expressed in the same cell should not be overlooked. Although these findings are novel to the field of nucleotide receptors, they confirm similar observations attained with other G-protein coupled receptors, emphasizing the dynamics and versatility of Gprotein coupled receptors in cell membranes and their signalling mechanism(s). Interestingly, the oligomers may have distinct physiological and pharmacological characteristics giving rise to novel properties, as has been described for the dimers of the multiple opioid receptor subunits which compose the μ, δ and the κ opioid receptor subtypes. The latter area is of obvious interest to the field of pain transduction and drug seeking behaviours, where these receptors and their ligands play a primordial role. On a structural basis, the dimerization process further emphasizes structural characteristics; the last 19 C-terminal amino acid residues play a critical role in oligomerization. Therefore, it is likely that the Ccarboxy ends of both P2Y1Rs intertwine allowing a more stable dimer complex, offering an image of how dimerization might occur in vivo and on the possible interactions with the G-proteins to which these receptors are coupled. Another issue addressed by this work relates to the relationship between agonist-induced dimerization and receptor desensitization. Clearly these are distinct events, although both are concentration-dependent; the authors discussed the hypothesis that dimerization is a prerequisite for internalization, independent of the interim pathway followed. In support of this possibility, the receptor mutant lacking the C-terminal 19 amino acid residues neither dimerized nor internalized. The authors additionally discuss that under pathophysiological conditions, cells may be exposed and encounter higher than physiological agonist levels, a situation that favours desensitization and the subsequent receptor internalization. A similar conclusion was reached by Norambuena et al., (Mol. Pharmacol. 74: 1666–1677, 2008) when studying the micro regionalization and internalization of the hP2Y1R from superficial placental vessels. They showed that ligand concentrations in the range of the receptor affinity exhibited almost null internalization within 30 min, while exposure of tissues to larger agonist concentrations accelerated markedly the internalization process within few minutes. Finally, the authors revealed that the P2Y1R cluster at a scaffold could also facilitate heterodimerization of the P2Y1R with interestingly potential binding partners such as the adenosine A1 or other receptors including another P2YR subtype.
Commentary Overall, this paper provides a strong biological basis for the understanding of P2Y1R functioning at the cellular and molecular level. The association of multiple receptor subunits to form functional channels for ligand-gated ionic channels or the requirement of nuclear or tyrosine kinase receptor dimerization as a prerequisite to initiate cell signalling is well established. However, G-protein coupled receptor oligomerization to form functional dimers was not always accepted, and was initially received with great scepticism. Before this paper, no information on putative P2YR dimers was available, emphasizing the impact of this study on the cell biology of these receptors and their contribution to purinegic signalling mechanism(s). Detection of P2Y1R dimers was achieved by the transfer of energy resonance between immediately adjacent fluorescently labelled proteins (FRET analysis); a sophisticated technique that requires ultra short distance interactions visualized through confocal microscopy in living cultured HEK 293 cells. The results indicate that a significant proportion of the P2Y1R in the cell membrane of these cells form homodimers, in a native resting state. Upon receptor activation with selective ligands, this fraction can be doubled, indicating that under this particular condition, almost all, if not all receptors dimerize. Since living cells are continually bombarded by multiple ligands, the possibility that these receptors form heterodimers with other nucleotide receptor subtypes or with non nucleotide
Gever J.R., Soto R., Henningsen R.A., Martin R.S., Hackos D.H., Panicker S., Rubas W., Oglesby I.B., Dillon M.P., Milla M.E., Burnstock, G., Ford, A.P.D.W (2010). AF-353, a novel, potent and orally bioavailable P2X3 P2X2/3 receptor antagonist. Br. J. Pharmacol., 160: 1387–1398. Article summary ATP is a neurotransmitter found primary afferents; dorsal horn neurons express P2X receptors either homotrimeric P2X2 or P2X3 and/or P2X2/3 heterotrimers which depolarize dorsal neurons activating the ascending pain pathway, which carries nociceptive information primarily to the brain stem, thalamus and hypothalamus. In view of the restricted expression of these P2X2 or P2X3R subtypes to sensory ganglion cells and their synapses, it has been hypothesized that these receptors could be targets for a novel family of analgesic drugs. This article describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3R antagonist. The potencies of AF-353 for the rat and human P2X3R and the human P2X2/3R were determined using radioligand binding methods, intracellular calcium flux and whole cell voltage-clamp electrophysiology. The pIC (50) estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of
J.P. Huidobro-Toro / Autonomic Neuroscience: Basic and Clinical 158 (2010) 1–4
receptors, enzymes and transporter proteins examined. In contrast to other compounds with a similar profile, the pharmacodynamics of AF353 is compatible with a non-competitive antagonist. Its pharmacokinetic parameters were overall favourable: oral bioavailability reached 32.9%, plasma protein binding amounted 98.2% and its plasma half-life was 90–100 min. The combination of a favourable pharmacokinetic profile together with its antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool for studying these channels in animal models. Commentary Putative antagonists acting on P2XR heterotrimers have long been sought with the aim of developing a new class of analgesic drugs, which could preclude the side effects of opioids or the COX inhibitors, the most popular antinociceptive agents used in and out of the hospital. AF-353 is a trimethoprim derivative with a promising profile as an orally active analgesic that partially overcomes the very severe pharmacokinetic limitations encountered by A-317491, the first P2X2/ 3R antagonist investigated early this decade. Among other restrictions of A-317491, which is not structurally related to trimethoprim, is that its oral bioavailability is extraordinarily low and in addition it has such an intense blood protein binding that its brain penetration is almost null. AF-353, in addition to its remarkable potency, shows a receptor selectivity profile for P2X2 and/or P2X2/3Rs. Although at this stage AF353 is far from reaching clinical trials, the hope is that this pharmacophore will lead to derivatives with markedly improved pharmacokinetic properties. Based on the primary afferent transmitter role of ATP, blocking P2X2/3R with lead compounds such as AF-353 must be considered a new strategy to combat pain. Therefore, this or a related derivative might, in addition to opioids and/or COX-inhibitors, form a new generation of rationally designed analgesic drugs, that might be used alone or in synergic combination with other analgesics. Therefore antinociceptive drugs operate through different mechanisms acting at peripheral sites like most COX inhibitors, the spinal cord, as in the case of opioids or P2X2/3R antagonists and central synapses as opioids and/or COX inhibitors.
Ase A.R., Bernier L.P., Blais D., Pankratov Y., Séguéla P. (2010). Modulation of heteromeric P2X1/5 receptors by phosphoinositides in astrocytes depends on the P2X1 subunit. J. Neurochem., 113: 1676–1684. Article summary Endogenous ATP-evoked currents with kinetics and pharmacological characteristics of the heteromeric P2X1/5 receptor channel have recently been reported. Little is known about the role of P2X receptors in astrocytes that only recently have been recognized to play an active role in modulating brain excitability. Therefore investigating P2X1R physiology and studying the role of phosphoinositides in astrocytes signalling is of great interest. Whole-cell patchclamp electrophysiological experiments were conducted in astrocytes isolated from cortical slices from a transgenic mouse model expressing enhanced green fluorescent protein-labelled astrocytes. Based on pharmacological studies examining the inhibition of the ATP-gated currents in astrocytes treated with wortmannin, an inhibitor of the enzyme phosphoinositide 3 kinase, the authors concluded that membrane phosphoinositides strongly modulate ATP-gated astrocyte P2X1/5-currents. Wortmannin selectively reduced both components of the P2X1/5R-gated currents without altering the kinetics of current recovery. These results were further examined in HEK 293 cell transfected with P2X1/5Rs, where the reduction of the ATP-gated current amplitude following wortmannin was rescued by the exogenous administration of PI(4,5)P2, but not by PI(3,4,5)P3, further
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indicating the specific role of PI(4,5)P2 on P2X1/5R signalling. Finally, in an attempt to identify the C-terminus amino acid residues and the of P2X5 subunit involved in phospholipid binding, the authors used an overlay receptor phosphoinositide binding assay to identify the positively charged amino acids in the 356 to 371 primary sequence of the of the P2X1R involved in PI(4,5)P(2) binding. Commentary In the past few years, several reports have shown that phosphoinositides modulate ionic channels; Séguéla's laboratory previously demonstrated the pivotal role of phosphoinositides for P2X1 and/or P2X4R activity, complementing a previous report on P2X2Rs. This article offers several novel findings that identify the P2X1/5R heteromer in astrocytes, adding unsuspected properties to these brain cells that had previously been regarded as passive partners or at best metabolic supporters of neurons. In addition, these authors demonstrated that phosphoinositide modulation of the heteromer resides only on the P2X1R subunit, rather than on both. The authors elegantly showed that the P2X5 subunit is insensitive to modulation by phosphoinositides, as shown by the electrophysiological recordings in cells expressing the P2X5R homomer construct and by the lack of binding of several anionic phosphoinositides to the proximal C-terminal domain of the P2X5 but not the P2X1R subunit. The latter is an ingenious assay system used in the Séguéla's lab to tentatively identify the binding site of the phosphoinositides to the C-terminal amino acid sequence that starts the second intracellular domain of these receptors. This latter finding allowed comparison of the amino acid residue sequence in the Cterminal end of the P2X1R subunit and tentatively identified those residues involved in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding. Although several basic amino residues are highly conserved in the 361–376 sequence of the P2XRs, the P2X5R has six basic amino acid residues in this sequence as opposed to seven in the P2X1R. It is as yet not clear which of the seven identified residues confers this property to the P2X channel. In previous reports, the authors performed sitedirected mutagenesis experiments to determine which of the positively charged amino acid residues in this receptor sequence conveys phosphoinositide binding (Bernier et al., J. Neurosci., 28: 12938–12945 (2008) and Bernier et al., Mol. Pharmacol., 74: 785–792 (2008)). In addition, using transfected HEK 293 cells with either the P2X1 and/or the P2X5Rs or the heteromer, the authors provide evidence that depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] levels using wortmannin, a PI-3 kinase inhibitor, decreases ATP-gated P2X1/5 evoked currents. Moreover, the intracellular application of varying concentrations of pure PI(4,5)P2 completely rescued the reduced ATPgated currents mediated by P2X1/5R in cells treated with wortmannin, demonstrating the need of this particular phosphoinositide, ruling out the involvement of other phosphatidylinositol such as PI (3,4,5)P. Finally, this paper complements recent findings on the role of ATP in astrocytes as a trigger signal for calcium waves where the activation of P2X and P2Y receptors is critical. Astrocytes are endowed with sets of functional P2X and P2Y receptors, both of which participate in intracellular Ca2+ mobilization albeit by different mechanisms. This finding indicates that ATP, acting through multiple membrane receptors, plays a prominent role in astroglial Ca 2+ signalling, in agreement with the notion that astrocytes display a specific form of excitability based on transient intracellular Ca2+ elevations, rather than neurons which rely on sodium and potassium transients to conduct action potentials.
Zemkova H., Kucka M., Li S., Gonzalez-Iglesias A.E., Tomic M., Stojilkovic S.S. (2010). Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells. Am. J. Physiol. Endocrinol. Metab., 298: E644-E651.
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Article summary Several lines of evidence have suggested that anterior pituitary cells release ATP and that the nucleotide might be involved in hormone secretion. Although the role of P2Y receptors had been partially addressed, little was known on the role of ATP-activated channels. This paper investigated which P2X channels are associated with anterior pituitary cells responsive to thyrotropin-releasing hormone. The most abundant mRNA transcripts in rat anterior pituitary cells correspond to the P2X4 subunit, a result confirmed by Western blot analysis of the P2X4R protein. Extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a concentration-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltageinsensitive Ca2+ influx. In the presence of ivermectin, a four-fold increase in the maximum ATP-induced inward current amplitude was noted, accompanied by an increase in the sensitivity of ATP receptors, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. In summary, pituitary cells like thyrotropin-releasing hormone-responsive and also lactotrophs express homomeric and/or heteromeric P2X4Rs, which facilitate Ca2+ influx and influence hormone secretion. Commentary That ATP plays a role in the regulation of anterior pituitary function was described almost 15 years ago, however, in situ determination and the P2X subtype involved in this tissue was absent. Using isolated pituitary cells from postpubertal female rats, the authors used a classical physiological approach, combining electrophysiology, cell biology and hormone secretion techniques to undertake the identification and characterization of the nucleotide P2XR subtype involved in anterior pituitary cell control. To accomplish this endeavour, authors combined a physiological oriented reasoning with cell biology and electrophysiological studies. The in situ identification of P2X4Rs in thyrotropin-releasing hormone-responsive cells was deduced from the large abundance of the mRNA for this receptor subtype. However, since the anterior pituitary cells expressed multiple P2X receptor subtypes, a functional role cannot be discarded for P2X4Rs heterotrimers; this issue remains open and is of course quite challenging because of the inherent difficulties of working with animal tissues. Most importantly, functional electrophysiological recordings complemented the cell characterization based on mRNA levels by the use of ATP concentration–response protocols performed in the absence and in the presence of IVM. This drug is a selective P2X4R allosteric modulator, which does not alter the ATP-gated currents in other P2XR subtype. Electrophysiological studies demonstrated that the ATP-gated concentration–response curve was displaced to the left, an unequivocal demonstration of the involvement of the P2X4R subtype in thyrotrophs. Although at present the exact mechanism of allosteric modulation by IVM has not been completely detailed, P2X4R IVM binding is presumably related to site(s) in the transmembrane receptor domain (Silberger et al., Neuron 54, 263–274, 2007). IVM is at present, together with trace metals, a reliable and specific tool that has proved valuable in P2XR characterization in whole tissues, as nicely illustrated in this study.
To further extend the study to other anterior pituitary cells, the authors analyzed the putative contribution of P2X4Rs to lactotrophs and prolactin secretion. An elegant experiment was designed: lactotrophs were challenged with ATP in the presence of variable concentrations of IVM and the output of prolactin was quantified. A concentration-dependent increase in the prolactin secretion peak amplitude and hormone release plateau was observed in cells challenged with ATP in the presence of 1–10 μM IVM, demonstrating that these cells also unequivocally express functional P2X4Rs which are coupled to active hormone secretion. The sequence of events that link receptor activation to hormone secretion is as follows. The influx of intracellular Ca2+ following P2X4R channel pore opening after ATP challenge initiates action potential firing in quiescent cells which increases cell firing frequency. Increased firing leads to voltagesensitive Ca2+ influx which finally results in prolactin secretion. The involvement of voltage dependent Ca2+ channels was deduced by the use of 1 μM nifedipine, a clinically relevant voltage-dependent calcium blocker. Since ATP is released from pituitary cells, the authors raise in addition, the possibility that vesicular-stored ATP stimulates the tissue P2X4 and P2Y1 receptors. The hypothesis was raised that nucleotide hydrolysis by tissue ectonucleotidases leads to ADP turning off P2X4R activity while prolonging P2Y1R signalling. Moreover, the further breakdown of ADP should result in adenosine, which might subsequently activate one or more of the several adenosine receptors expressed in anterior pituitary cells. Adenosine is known to inhibit neuronal activity, silencing brain excitability; a mechanism that provides insights into the versatility of purinergic signalling in central and peripheral synapses, and at neuroeffector junctions. Concluding remarks Nucleotide receptors, belonging to either the P2X or P2Y family, have commonalities with other ionotropic or G-protein coupled receptors, independent of the uniqueness of each receptor primary amino acid sequence and their expression in multiple cell systems in excitable and non-excitable cell and tissues. The finding that subunits of the P2X receptors oligomerize into different heterotrimers speaks of their functional versatility, similar to pentameric receptors of nicotinic, 5-HT3 or GABAA subtypes. The in situ identification of each subunit expression to confirm the multiple heterotrimers remains a challenge. The crystallization of the P2X4R is providing opportunities for the molecular understanding of channel gating, allosteric modulation and will also guide a rational basis for drug design as documented for the P2X2/3Rs and the development of novel analgesics. A similar commentary applies for the dimerization or even higher forms of P2Y receptors, oligomerization, an evolving theme that will provide new vistas concerning signalling in health and disease. Knowledge of the principles of nucleotide receptor cell biology will improve our appreciation of receptor recycling and internalization, desensitization, post-translational modifications, receptor trafficking, cell surface distribution and finally modulation of each subunit expression by physiological and pathophysiological stimuli. Future challenges are to further define and identify other critical structural elements of the P2XRs and P2YRs. Role of funding source Funding: PFB 12/2007.
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
HIGHLIGHTS IN BASIC AUTONOMIC NEUROSCIENCES: NUCLEOTIDE RECEPTORS: MOLECULAR SIGNALLING AND STRUCTURAL ELEMENTS Prepared by: J. Pablo Huidobro-Toro Nucleotide Research Labs, Department of Physiology, Faculty of Biological Sciences, P. Catholic University of Chile, Casilla 114-D, Santiago, Chile Section Editor: Michael P. Gilbey
Extracellular nucleotides such as ATP, UTP, ADP and UDP have attracted much attention as novel messengers of non-excitable and excitable cells, including peripheral autonomic and central nerve endings. Nucleotides are released into the extracellular milieu by a variety of mechanisms involving channels, transporters and synaptic vesicles; these molecules act as auto/paracrine signals. A set of at least 15 plasma membrane receptors respond to extracellular nucleotides and belong to either the ATP-gated ionic channels, which form either homo- or heteromeric receptors (P2XR1–7 subtypes), or are G-protein coupled (eight clones of P2YR receptor subtypes), most of these receptors oligomerize. These receptors do not require γ phosphate hydrolysis to signal intracellularly and are involved in many autonomic control functions including those of blood flow, bladder voiding, intestinal motility and secretion, and airway conductance.
Kawate T., Michel J.C., Birdsong, W., Gouaux, E. (2009). Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460: 592–598. Article summary P2X receptors are a novel family of cation-selective ion channels gated by extracellular ATP. Because P2X receptors are not related to other ion channel proteins of known structure, it was a challenge to crystallize a representative family member and examine the crystals by X rays to decipher its 3-D structure. To improve crystallization, the authors took advantage of zebrafish P2X4R deletion mutants lacking both the N and C terminus intracellular domains; mutants maintained unaltered the complete extracellular and transmembrane domains. The chalice-shaped trimeric receptor is composed of three entangled subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in beta-strands, have large acidic patches that may attract cations through fenestrations to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an approximately 8 Å slab of protein. The location of three non-canonical, intersubunit ATP-binding sites was defined, suggesting that ATP binding promotes subunit rearrangement and ion 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.08.001
channel opening. The orthosteric ATP binding site is not in the immediate vicinity of the receptor transmembrane domain. Commentary The crystal structure of this ATP-gated P2X ion channel was analyzed in its close, resting state, with a 3.1 Å resolution; it provided unequivocal evidence, at an atomic scale, of the trimetric subunit channel stoichiometry, confirming prior empirical biochemical analysis. The authors compare the 3-D receptor contour of a single subunit to a dolphin-like shape. They reveal with precision, the 3-D organization of the large extracellular domain of each subunit and the interfaces between adjacent subunits. The putative orthosteric site for ATP binding was tentatively localized in the outer part of the extracellular domain; likely nucleotide binding induces conformational changes within and between subunits such that the pore topology accommodates for cation influx. The exact spatial location of the 12 highly conserved extracellular cysteines was revealed and which of these residues form each of the six predicted S–S bond pairs of the extracellular domain was determined. The level of resolution permitted the visualization of the 3-D intersubunit contacts required to form the channel pore independent of homo- or heteromer subunit assembly. The 3-D viewing allowed an insight into the pore entry identifying an upper, central and an extracellular vestibule in the extracellular subunit domain linked to the intracellular vestibule explaining how the ions permeate the cell. The channel pore is located on the molecular three-fold axis, with the extracellular vestibule connected to the bulk solution through a fenestration, allowing the entrance of cations. The proposed P2X channel receptor model allowed the further use of other bioinformatics programs to further investigate and identify through modelling the putative spatial localization of trace metal allosteric modulator sites which had been proposed to either increase or decrease the receptor affinity for ATP. Surprisingly when the reported amino acids suspected to conform part of the trace metal coordination site were identified and visualized in the P2X4R 3-D structure, they were found to be located in the immediate vicinity of the purported ATP binding site, in agreement with Coddou et al. (J. Biol. Chem., 282: 36879–36886, 2007). However, and far more importantly, the structural backbone of the P2X4R will allow the modelling of the complete P2X receptors, a necessary first
2
J.P. Huidobro-Toro / Autonomic Neuroscience: Basic and Clinical 158 (2010) 1–4
step towards the goal of rational drug design, and will provide essential data required to understand the details of pore opening kinetics and explain the electrophysiological properties of each channel subtypes and their fine intricacies at the molecular level. This paper is a classic in the field due to its three-dimensional visualization of the P2X4R conformation.
Choi R.C.Y., Simon J., Tsim K.W.K. Barnard E.A. (2008). Constitutive and agonist-induced dimerization of the P2Y1 receptor: relationship to internalization and scaffolding. J. Biol. Chem., 283: 11050–11063. Article summary Although initially visualized as independent cell membrane proteins, G-protein coupled receptors are now generally thought as dimers forming either homo- or heterooligomers. This study was the first to address the formation of P2Y receptor oligomers and assessed whether selective receptor activation with preferential ligands modifies the state of receptor dimerization. In living cells, P2Y1R dimerization was quantified by an improved version of fluorescence resonance energy transfer donor photobleaching examination. In the resting state, 44% of the P2Y1Rs expressed in HEK293 cell membranes are dimers; however, agonist exposure induced up to 85–100% dimerization. Monomer and constitutive dimers are fully active in cell signalling. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonists, an indication of a reversible process. Receptor dimers are required for internalization of the P2Y1R at 37 °C; at 20 °C dimerization also occurs, but endocytosis is abolished. Removal of the Cterminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. Distinction should therefore be made between 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signalling membrane micro-domain, and 2) free dimers in the cell membrane, which may dimerize by agonist exposure. In summary, for class A G-protein coupled receptors, the authors suggest that the percentages of free monomers, although variable, may oscillate up to 40%; agonist induced dimers are variable and may amount to as much as 100% of the cell membrane receptor population.
receptors expressed in the same cell should not be overlooked. Although these findings are novel to the field of nucleotide receptors, they confirm similar observations attained with other G-protein coupled receptors, emphasizing the dynamics and versatility of Gprotein coupled receptors in cell membranes and their signalling mechanism(s). Interestingly, the oligomers may have distinct physiological and pharmacological characteristics giving rise to novel properties, as has been described for the dimers of the multiple opioid receptor subunits which compose the μ, δ and the κ opioid receptor subtypes. The latter area is of obvious interest to the field of pain transduction and drug seeking behaviours, where these receptors and their ligands play a primordial role. On a structural basis, the dimerization process further emphasizes structural characteristics; the last 19 C-terminal amino acid residues play a critical role in oligomerization. Therefore, it is likely that the Ccarboxy ends of both P2Y1Rs intertwine allowing a more stable dimer complex, offering an image of how dimerization might occur in vivo and on the possible interactions with the G-proteins to which these receptors are coupled. Another issue addressed by this work relates to the relationship between agonist-induced dimerization and receptor desensitization. Clearly these are distinct events, although both are concentration-dependent; the authors discussed the hypothesis that dimerization is a prerequisite for internalization, independent of the interim pathway followed. In support of this possibility, the receptor mutant lacking the C-terminal 19 amino acid residues neither dimerized nor internalized. The authors additionally discuss that under pathophysiological conditions, cells may be exposed and encounter higher than physiological agonist levels, a situation that favours desensitization and the subsequent receptor internalization. A similar conclusion was reached by Norambuena et al., (Mol. Pharmacol. 74: 1666–1677, 2008) when studying the micro regionalization and internalization of the hP2Y1R from superficial placental vessels. They showed that ligand concentrations in the range of the receptor affinity exhibited almost null internalization within 30 min, while exposure of tissues to larger agonist concentrations accelerated markedly the internalization process within few minutes. Finally, the authors revealed that the P2Y1R cluster at a scaffold could also facilitate heterodimerization of the P2Y1R with interestingly potential binding partners such as the adenosine A1 or other receptors including another P2YR subtype.
Commentary Overall, this paper provides a strong biological basis for the understanding of P2Y1R functioning at the cellular and molecular level. The association of multiple receptor subunits to form functional channels for ligand-gated ionic channels or the requirement of nuclear or tyrosine kinase receptor dimerization as a prerequisite to initiate cell signalling is well established. However, G-protein coupled receptor oligomerization to form functional dimers was not always accepted, and was initially received with great scepticism. Before this paper, no information on putative P2YR dimers was available, emphasizing the impact of this study on the cell biology of these receptors and their contribution to purinegic signalling mechanism(s). Detection of P2Y1R dimers was achieved by the transfer of energy resonance between immediately adjacent fluorescently labelled proteins (FRET analysis); a sophisticated technique that requires ultra short distance interactions visualized through confocal microscopy in living cultured HEK 293 cells. The results indicate that a significant proportion of the P2Y1R in the cell membrane of these cells form homodimers, in a native resting state. Upon receptor activation with selective ligands, this fraction can be doubled, indicating that under this particular condition, almost all, if not all receptors dimerize. Since living cells are continually bombarded by multiple ligands, the possibility that these receptors form heterodimers with other nucleotide receptor subtypes or with non nucleotide
Gever J.R., Soto R., Henningsen R.A., Martin R.S., Hackos D.H., Panicker S., Rubas W., Oglesby I.B., Dillon M.P., Milla M.E., Burnstock, G., Ford, A.P.D.W (2010). AF-353, a novel, potent and orally bioavailable P2X3 P2X2/3 receptor antagonist. Br. J. Pharmacol., 160: 1387–1398. Article summary ATP is a neurotransmitter found primary afferents; dorsal horn neurons express P2X receptors either homotrimeric P2X2 or P2X3 and/or P2X2/3 heterotrimers which depolarize dorsal neurons activating the ascending pain pathway, which carries nociceptive information primarily to the brain stem, thalamus and hypothalamus. In view of the restricted expression of these P2X2 or P2X3R subtypes to sensory ganglion cells and their synapses, it has been hypothesized that these receptors could be targets for a novel family of analgesic drugs. This article describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3R antagonist. The potencies of AF-353 for the rat and human P2X3R and the human P2X2/3R were determined using radioligand binding methods, intracellular calcium flux and whole cell voltage-clamp electrophysiology. The pIC (50) estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of
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receptors, enzymes and transporter proteins examined. In contrast to other compounds with a similar profile, the pharmacodynamics of AF353 is compatible with a non-competitive antagonist. Its pharmacokinetic parameters were overall favourable: oral bioavailability reached 32.9%, plasma protein binding amounted 98.2% and its plasma half-life was 90–100 min. The combination of a favourable pharmacokinetic profile together with its antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool for studying these channels in animal models. Commentary Putative antagonists acting on P2XR heterotrimers have long been sought with the aim of developing a new class of analgesic drugs, which could preclude the side effects of opioids or the COX inhibitors, the most popular antinociceptive agents used in and out of the hospital. AF-353 is a trimethoprim derivative with a promising profile as an orally active analgesic that partially overcomes the very severe pharmacokinetic limitations encountered by A-317491, the first P2X2/ 3R antagonist investigated early this decade. Among other restrictions of A-317491, which is not structurally related to trimethoprim, is that its oral bioavailability is extraordinarily low and in addition it has such an intense blood protein binding that its brain penetration is almost null. AF-353, in addition to its remarkable potency, shows a receptor selectivity profile for P2X2 and/or P2X2/3Rs. Although at this stage AF353 is far from reaching clinical trials, the hope is that this pharmacophore will lead to derivatives with markedly improved pharmacokinetic properties. Based on the primary afferent transmitter role of ATP, blocking P2X2/3R with lead compounds such as AF-353 must be considered a new strategy to combat pain. Therefore, this or a related derivative might, in addition to opioids and/or COX-inhibitors, form a new generation of rationally designed analgesic drugs, that might be used alone or in synergic combination with other analgesics. Therefore antinociceptive drugs operate through different mechanisms acting at peripheral sites like most COX inhibitors, the spinal cord, as in the case of opioids or P2X2/3R antagonists and central synapses as opioids and/or COX inhibitors.
Ase A.R., Bernier L.P., Blais D., Pankratov Y., Séguéla P. (2010). Modulation of heteromeric P2X1/5 receptors by phosphoinositides in astrocytes depends on the P2X1 subunit. J. Neurochem., 113: 1676–1684. Article summary Endogenous ATP-evoked currents with kinetics and pharmacological characteristics of the heteromeric P2X1/5 receptor channel have recently been reported. Little is known about the role of P2X receptors in astrocytes that only recently have been recognized to play an active role in modulating brain excitability. Therefore investigating P2X1R physiology and studying the role of phosphoinositides in astrocytes signalling is of great interest. Whole-cell patchclamp electrophysiological experiments were conducted in astrocytes isolated from cortical slices from a transgenic mouse model expressing enhanced green fluorescent protein-labelled astrocytes. Based on pharmacological studies examining the inhibition of the ATP-gated currents in astrocytes treated with wortmannin, an inhibitor of the enzyme phosphoinositide 3 kinase, the authors concluded that membrane phosphoinositides strongly modulate ATP-gated astrocyte P2X1/5-currents. Wortmannin selectively reduced both components of the P2X1/5R-gated currents without altering the kinetics of current recovery. These results were further examined in HEK 293 cell transfected with P2X1/5Rs, where the reduction of the ATP-gated current amplitude following wortmannin was rescued by the exogenous administration of PI(4,5)P2, but not by PI(3,4,5)P3, further
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indicating the specific role of PI(4,5)P2 on P2X1/5R signalling. Finally, in an attempt to identify the C-terminus amino acid residues and the of P2X5 subunit involved in phospholipid binding, the authors used an overlay receptor phosphoinositide binding assay to identify the positively charged amino acids in the 356 to 371 primary sequence of the of the P2X1R involved in PI(4,5)P(2) binding. Commentary In the past few years, several reports have shown that phosphoinositides modulate ionic channels; Séguéla's laboratory previously demonstrated the pivotal role of phosphoinositides for P2X1 and/or P2X4R activity, complementing a previous report on P2X2Rs. This article offers several novel findings that identify the P2X1/5R heteromer in astrocytes, adding unsuspected properties to these brain cells that had previously been regarded as passive partners or at best metabolic supporters of neurons. In addition, these authors demonstrated that phosphoinositide modulation of the heteromer resides only on the P2X1R subunit, rather than on both. The authors elegantly showed that the P2X5 subunit is insensitive to modulation by phosphoinositides, as shown by the electrophysiological recordings in cells expressing the P2X5R homomer construct and by the lack of binding of several anionic phosphoinositides to the proximal C-terminal domain of the P2X5 but not the P2X1R subunit. The latter is an ingenious assay system used in the Séguéla's lab to tentatively identify the binding site of the phosphoinositides to the C-terminal amino acid sequence that starts the second intracellular domain of these receptors. This latter finding allowed comparison of the amino acid residue sequence in the Cterminal end of the P2X1R subunit and tentatively identified those residues involved in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding. Although several basic amino residues are highly conserved in the 361–376 sequence of the P2XRs, the P2X5R has six basic amino acid residues in this sequence as opposed to seven in the P2X1R. It is as yet not clear which of the seven identified residues confers this property to the P2X channel. In previous reports, the authors performed sitedirected mutagenesis experiments to determine which of the positively charged amino acid residues in this receptor sequence conveys phosphoinositide binding (Bernier et al., J. Neurosci., 28: 12938–12945 (2008) and Bernier et al., Mol. Pharmacol., 74: 785–792 (2008)). In addition, using transfected HEK 293 cells with either the P2X1 and/or the P2X5Rs or the heteromer, the authors provide evidence that depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] levels using wortmannin, a PI-3 kinase inhibitor, decreases ATP-gated P2X1/5 evoked currents. Moreover, the intracellular application of varying concentrations of pure PI(4,5)P2 completely rescued the reduced ATPgated currents mediated by P2X1/5R in cells treated with wortmannin, demonstrating the need of this particular phosphoinositide, ruling out the involvement of other phosphatidylinositol such as PI (3,4,5)P. Finally, this paper complements recent findings on the role of ATP in astrocytes as a trigger signal for calcium waves where the activation of P2X and P2Y receptors is critical. Astrocytes are endowed with sets of functional P2X and P2Y receptors, both of which participate in intracellular Ca2+ mobilization albeit by different mechanisms. This finding indicates that ATP, acting through multiple membrane receptors, plays a prominent role in astroglial Ca 2+ signalling, in agreement with the notion that astrocytes display a specific form of excitability based on transient intracellular Ca2+ elevations, rather than neurons which rely on sodium and potassium transients to conduct action potentials.
Zemkova H., Kucka M., Li S., Gonzalez-Iglesias A.E., Tomic M., Stojilkovic S.S. (2010). Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells. Am. J. Physiol. Endocrinol. Metab., 298: E644-E651.
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Article summary Several lines of evidence have suggested that anterior pituitary cells release ATP and that the nucleotide might be involved in hormone secretion. Although the role of P2Y receptors had been partially addressed, little was known on the role of ATP-activated channels. This paper investigated which P2X channels are associated with anterior pituitary cells responsive to thyrotropin-releasing hormone. The most abundant mRNA transcripts in rat anterior pituitary cells correspond to the P2X4 subunit, a result confirmed by Western blot analysis of the P2X4R protein. Extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a concentration-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltageinsensitive Ca2+ influx. In the presence of ivermectin, a four-fold increase in the maximum ATP-induced inward current amplitude was noted, accompanied by an increase in the sensitivity of ATP receptors, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. In summary, pituitary cells like thyrotropin-releasing hormone-responsive and also lactotrophs express homomeric and/or heteromeric P2X4Rs, which facilitate Ca2+ influx and influence hormone secretion. Commentary That ATP plays a role in the regulation of anterior pituitary function was described almost 15 years ago, however, in situ determination and the P2X subtype involved in this tissue was absent. Using isolated pituitary cells from postpubertal female rats, the authors used a classical physiological approach, combining electrophysiology, cell biology and hormone secretion techniques to undertake the identification and characterization of the nucleotide P2XR subtype involved in anterior pituitary cell control. To accomplish this endeavour, authors combined a physiological oriented reasoning with cell biology and electrophysiological studies. The in situ identification of P2X4Rs in thyrotropin-releasing hormone-responsive cells was deduced from the large abundance of the mRNA for this receptor subtype. However, since the anterior pituitary cells expressed multiple P2X receptor subtypes, a functional role cannot be discarded for P2X4Rs heterotrimers; this issue remains open and is of course quite challenging because of the inherent difficulties of working with animal tissues. Most importantly, functional electrophysiological recordings complemented the cell characterization based on mRNA levels by the use of ATP concentration–response protocols performed in the absence and in the presence of IVM. This drug is a selective P2X4R allosteric modulator, which does not alter the ATP-gated currents in other P2XR subtype. Electrophysiological studies demonstrated that the ATP-gated concentration–response curve was displaced to the left, an unequivocal demonstration of the involvement of the P2X4R subtype in thyrotrophs. Although at present the exact mechanism of allosteric modulation by IVM has not been completely detailed, P2X4R IVM binding is presumably related to site(s) in the transmembrane receptor domain (Silberger et al., Neuron 54, 263–274, 2007). IVM is at present, together with trace metals, a reliable and specific tool that has proved valuable in P2XR characterization in whole tissues, as nicely illustrated in this study.
To further extend the study to other anterior pituitary cells, the authors analyzed the putative contribution of P2X4Rs to lactotrophs and prolactin secretion. An elegant experiment was designed: lactotrophs were challenged with ATP in the presence of variable concentrations of IVM and the output of prolactin was quantified. A concentration-dependent increase in the prolactin secretion peak amplitude and hormone release plateau was observed in cells challenged with ATP in the presence of 1–10 μM IVM, demonstrating that these cells also unequivocally express functional P2X4Rs which are coupled to active hormone secretion. The sequence of events that link receptor activation to hormone secretion is as follows. The influx of intracellular Ca2+ following P2X4R channel pore opening after ATP challenge initiates action potential firing in quiescent cells which increases cell firing frequency. Increased firing leads to voltagesensitive Ca2+ influx which finally results in prolactin secretion. The involvement of voltage dependent Ca2+ channels was deduced by the use of 1 μM nifedipine, a clinically relevant voltage-dependent calcium blocker. Since ATP is released from pituitary cells, the authors raise in addition, the possibility that vesicular-stored ATP stimulates the tissue P2X4 and P2Y1 receptors. The hypothesis was raised that nucleotide hydrolysis by tissue ectonucleotidases leads to ADP turning off P2X4R activity while prolonging P2Y1R signalling. Moreover, the further breakdown of ADP should result in adenosine, which might subsequently activate one or more of the several adenosine receptors expressed in anterior pituitary cells. Adenosine is known to inhibit neuronal activity, silencing brain excitability; a mechanism that provides insights into the versatility of purinergic signalling in central and peripheral synapses, and at neuroeffector junctions. Concluding remarks Nucleotide receptors, belonging to either the P2X or P2Y family, have commonalities with other ionotropic or G-protein coupled receptors, independent of the uniqueness of each receptor primary amino acid sequence and their expression in multiple cell systems in excitable and non-excitable cell and tissues. The finding that subunits of the P2X receptors oligomerize into different heterotrimers speaks of their functional versatility, similar to pentameric receptors of nicotinic, 5-HT3 or GABAA subtypes. The in situ identification of each subunit expression to confirm the multiple heterotrimers remains a challenge. The crystallization of the P2X4R is providing opportunities for the molecular understanding of channel gating, allosteric modulation and will also guide a rational basis for drug design as documented for the P2X2/3Rs and the development of novel analgesics. A similar commentary applies for the dimerization or even higher forms of P2Y receptors, oligomerization, an evolving theme that will provide new vistas concerning signalling in health and disease. Knowledge of the principles of nucleotide receptor cell biology will improve our appreciation of receptor recycling and internalization, desensitization, post-translational modifications, receptor trafficking, cell surface distribution and finally modulation of each subunit expression by physiological and pathophysiological stimuli. Future challenges are to further define and identify other critical structural elements of the P2XRs and P2YRs. Role of funding source Funding: PFB 12/2007.