RESUMO
P2X receptors are ATP-gated trimeric nonselective cation channels that are important for various physiological and pathological processes, including synaptic transmission, pain perception, immune regulation, and apoptosis. Accordingly, they attract a wide range of interest as drug targets, such as those for chronic cough, neuropathic pain, and depression. After the zebrafish P2X4 receptor structure was reported in 2009, various other P2X receptor structures have been reported, extending our understanding of the molecular mechanisms of P2X receptors. This review article describes the recent progress on understanding the structures and mechanisms of P2X receptors, especially of the mechanisms underlying ATP binding and conformational changes during the gating cycle. In addition, since several antagonists for different P2X subtypes have entered into clinical trials, this review also summarizes the binding sites and regulatory mechanisms of these antagonists, which may contribute to new strategies of targeting P2X receptors for drug discovery.
Assuntos
Receptores Purinérgicos P2X4 , Peixe-Zebra , Trifosfato de Adenosina/química , Animais , Biologia , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/metabolismo , Peixe-Zebra/metabolismoRESUMO
Since the X-ray structure of the zebra fish P2X4 receptor in the closed state was published in 2009 homology modeling has been used to generate structural models for P2X receptors. In this chapter, we outline how to use the MODELLER software to generate such structural models for P2X receptors whose structures have not been solved yet.
Assuntos
Receptores Purinérgicos P2X/química , Software , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Animais , Humanos , Homologia de SequênciaRESUMO
Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein-protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/metabolismo , Humanos , Medições Luminescentes , Ligação Proteica , Transdução de SinaisRESUMO
Myocardial ischemia evokes powerful reflex responses through activation of vagal and sympathetic afferents in the heart through the release of ischemic metabolites. We have demonstrated that extracellular ATP stimulates cardiac sympathetic afferents through P2 receptor-mediated mechanism, and that opioid peptides suppress these afferents' activity. However, the roles of both P2 receptor and endogenous opioids in cardiac sympathoexcitatory reflex (CSR) responses remain unclear. We therefore hypothesized that activation of cardiac P2 receptor evokes CSR responses by stimulating cardiac sympathetic afferents and these CSR responses are modulated by endogenous opioids. We observed that intrapericardial injection of α,ß-methylene ATP (α,ß-meATP, P2X receptor agonist), but not ADP (P2Y receptor agonist), caused a graded increase in mean arterial pressure in rats with sinoaortic denervation and vagotomy. This effect of α,ß-meATP was abolished by blockade of cardiac neural transmission with intrapericardial procaine treatment and eliminated by intrapericardial A-317491, a selective P2X2/3 and P2X3 receptor antagonist. Intrapericardial α,ß-meATP also evoked CSR response in vagus-intact rats. Furthermore, the P2X receptor-mediated CSR responses were enhanced by intrapericardial naloxone, a specific opioid receptor antagonist. These data suggest that stimulation of cardiac P2X2/3 and P2X3, but not P2Y receptors, powerfully evokes CSR responses through activation of cardiac spinal afferents, and that endogenous opioids suppress the P2X receptor-mediated CSR responses.
Assuntos
Coração/fisiologia , Receptores Opioides/metabolismo , Receptores Purinérgicos P2X/metabolismo , Reflexo/fisiologia , Sistema Nervoso Simpático/fisiologia , Anestésicos Locais/farmacologia , Animais , Regulação da Expressão Gênica , Coração/efeitos dos fármacos , Masculino , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Procaína/farmacologia , Agonistas do Receptor Purinérgico P2/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides/genética , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/genética , Reflexo/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: The atrium is exposed to high shear stress during heart failure and valvular diseases. We aimed to understand atrial shear-induced Ca2+ signaling and its underlying mechanisms. METHODS: Pressurized micro-flow was applied to single rat atrial myocytes, and Ca2+ signal, membrane potential, and ATP release were assessed using confocal imaging, patch clamp technique, and luciferin-luciferase assay, respectively. RESULTS: Shear stress (â¼16 dyn/cm2) induced global Ca2+ waves (â¼0.1 events/s) from the periphery to the center of cells in a transverse direction ("T-wave"; â¼145 µm/s). Pharmacological interventions and simultaneous recording of membrane potential and Ca2+ demonstrated that shear-induced T-waves resulted from action potential (AP)-triggered Ca2+ release from the sarcoplasmic reticulum. T-waves were not sensitive to inhibitors of known shear signaling mechanisms except connexin hemichannels and ATP release. Shear stress caused ATP release from these myocytes (â¼1.1x10-17 moles/unit membrane, µm2); ATP release was increased by enhancement of connexin hemichannels and suppressed by inhibition of the hemichannels, but not affected by inhibitors of other ATP release pathways. Blockade of P2X receptor, but not pannexin or the Na+-Ca2+ exchanger, eliminated shear-induced T-wave initiation. CONCLUSION: Our data suggest that shear stress triggers APs and concomitant Ca2+ signaling via activation of P2X receptors by connexin hemichannel-mediated ATP release in atrial myocytes.
Assuntos
Sinalização do Cálcio , Receptores Purinérgicos P2X/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Conexinas/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , NADPH Oxidases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X/química , Retículo Sarcoplasmático/metabolismo , Resistência ao Cisalhamento , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Tetrodotoxina/farmacologiaRESUMO
The P2X receptor is a trimeric transmembrane protein that acts as an ATP-gated ion channel. Its transmembrane domain (TMD) contains only six helices and three of them, the M2 helices, line the ion conduction pathway. Here, using molecular dynamics simulation, I identify four conformational states of the TMD that are associated with four types of packing between M2 helices. Packing in the extracellular half of the M2 helix produces closed conformations, while packing in the intracellular half produces both open and closed conformations. State transition is observed and supports a mechanism where iris-like twisting of the M2 helices switches the location of helical packing between the extracellular and the intracellular halves of the helices. In addition, this twisting motion alters the position and orientation of residue side-chains relative to the pore and therefore influences the pore geometry and possibly ion permeation. Helical packing, on the other hand, may restrict the twisting motion and generate discrete conformational states.
Assuntos
Membrana Celular/química , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores Purinérgicos P2X/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Domínios ProteicosRESUMO
Multi-target-directed ligands (MTDLs) offer new hope for the treatment of multifactorial complex diseases such as Alzheimer's Disease (AD). Herein, we present compounds aimed at targeting the NMDA and the P2X7 receptors, which embody a different approach to AD therapy. On one hand, we are seeking to delay neurodegeneration targeting the glutamatergic NMDA receptors; on the other hand, we also aim to reduce neuroinflammation, targeting P2X7 receptors. Although the NMDA receptor is a widely recognized therapeutic target in treating AD, the P2X7 receptor remains largely unexplored for this purpose; therefore, the dual inhibitor presented herein-which is open to further optimization-represents the first member of a new class of MTDLs.
Assuntos
Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Linhagem Celular Tumoral , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Desenho de Fármacos , Descoberta de Drogas , Humanos , Concentração Inibidora 50 , Ligantes , Estrutura Molecular , Antagonistas do Receptor Purinérgico P2X/síntese química , Receptores Purinérgicos P2X/metabolismoRESUMO
P2X receptors are cation-selective channels that are activated by the binding of extracellular ATP. They have a high permeability to Ca2+, Na+, and K+ and are expressed widely throughout the nervous, immune, cardiovascular, skeletal, gastrointestinal, respiratory, and endocrine systems. Seven mammalian subtypes of P2X receptor subunits have been identified, P2X1-7, and those that function as homotrimeric receptors (P2X1, 2, 3, 4, and 7) are targeted to lipid rafts, although they show limited resistance to solubilization by Triton X-100. Recent crystal structures of P2X3 and P2X4 receptors have provided considerable high-resolution information about the architecture of this family of receptors and yet the molecular details of how they are regulated by cholesterol are unknown. Currents mediated by the P2X1-4 receptors are either inhibited or relatively insensitive to cholesterol depletion, but there is no clear evidence to support the direct binding of cholesterol to these receptors. In contrast, the activity of the low-affinity, proinflammatory P2X7 receptor is potentiated by cholesterol depletion and regions within the proximal C-terminus play an important role in coupling changes in cholesterol to the gating of the pore. Based upon our understanding of the lipid signaling events that are triggered downstream of P2X7 receptor activation, a change in the levels of cholesterol may contribute to the sensitization of receptor currents and the dilation of the pore that occurs following prolonged, high-level stimulation. This chapter focuses on the regulation of P2X7 receptor signaling by cholesterol and our current understanding of the mechanisms that underlie this.
Assuntos
Colesterol/metabolismo , Receptores Purinérgicos P2X/metabolismo , Transdução de Sinais , Animais , Humanos , Microdomínios da Membrana/metabolismo , Receptores Purinérgicos P2X/químicaRESUMO
Extracellular ATP-gated P2X receptors are trimeric non-selective cation channels important for many physiological events including immune response and neural transmission. These receptors belong to a unique class of ligand-gated ion channels composed of only six transmembrane helices and a relatively small extracellular domain that harbors three ATP-binding pockets. The crystal structures of P2X receptors, including the recent P2X3 structures representing three different stages of the gating cycle, have provided a compelling structural foundation for understanding how this class of ligand-gated ion channels function. These structures, in combination with numerous functional studies ranging from classic mutagenesis and electrophysiology to modern optogenetic pharmacology, have uncovered unique molecular mechanisms of P2X receptor function. This review article summarizes the current knowledge in P2X receptor activation, especially focusing on the mechanisms underlying ATP-binding, conformational changes in the extracellular domain, and channel gating and desensitization.
Assuntos
Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2X , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Humanos , Domínios Proteicos , Relação Quantitativa Estrutura-Atividade , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/metabolismoRESUMO
P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors.
Assuntos
Citidina Trifosfato/metabolismo , Receptores Purinérgicos P2X/química , Proteínas de Peixe-Zebra/química , Animais , Sítios de Ligação , Citidina Trifosfato/química , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores Purinérgicos P2X/metabolismo , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na(+), K(+) and Ca(2+). Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors.
Assuntos
Receptores Purinérgicos P2X/fisiologia , Regulação Alostérica , Animais , Domínio Catalítico , Humanos , Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Domínios Proteicos , Receptores Purinérgicos P2X/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.
Assuntos
Proteínas de Artrópodes/química , Receptores Purinérgicos P2X/química , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Magnésio/farmacologia , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Receptores Purinérgicos P2X/metabolismo , Carrapatos , Xenopus , Zinco/farmacologiaRESUMO
ATP-gated P2X receptors are trimeric ion channels selective to cations. Recent progress in the molecular biophysics of these channels enables a better understanding of their function. In particular, data obtained from biochemical, electrophysiogical and molecular engineering in the light of recent X-ray structures now allow delineation of the principles of ligand binding, channel opening and allosteric modulation. However, although a picture emerges as to how ATP triggers channel opening, there are a number of intriguing questions that remain to be answered, in particular how the pore itself opens in response to ATP and how the intracellular domain, for which structural information is limited, moves during activation. In this review, we provide a summary of functional studies in the context of the post-structure era, aiming to clarify our understanding of the way in which P2X receptors function in response to ATP binding, as well as the mechanism by which allosteric modulators are able to regulate receptor function. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico , Receptores Purinérgicos P2X/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Humanos , Optogenética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Receptores Purinérgicos P2X/químicaRESUMO
The P2X purinergic receptors are cation-selective channels gated by extracellular adenosine 5'-triphosphate (ATP). These purinergic receptors are found in virtually all mammalian cell types and facilitate a number of important physiological processes. Within the past few years, the characterization of crystal structures of the zebrafish P2X4 receptor in its closed and open states has provided critical insights into the mechanisms of ligand binding and channel activation. Understanding of this gating mechanism has facilitated to design and interpret new modeling and structure-function experiments to better elucidate how different agonists and antagonists can affect the receptor with differing levels of potency. This review summarizes the current knowledge on the structure, activation, allosteric modulators, function, and location of the different P2X receptors. Moreover, an emphasis on the P2X2 receptors has been placed in respect to its role in the auditory system. In particular, the discovery of three missense mutations in P2X2 receptors could become important areas of study in the field of gene therapy to treat progressive and noise-induced hearing loss. J. Cell. Physiol. 231: 1656-1670, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Trifosfato de Adenosina/metabolismo , Vias Auditivas/metabolismo , Perda Auditiva Provocada por Ruído/metabolismo , Audição , Ativação do Canal Iônico , Receptores Purinérgicos P2X/metabolismo , Transdução de Sinais , Animais , Vias Auditivas/efeitos dos fármacos , Vias Auditivas/fisiopatologia , Predisposição Genética para Doença , Audição/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/genética , Perda Auditiva Provocada por Ruído/fisiopatologia , Perda Auditiva Provocada por Ruído/terapia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Conformação Proteica , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/efeitos dos fármacos , Receptores Purinérgicos P2X/genética , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain, diabetes, ischemic protection and many other conditions. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.
Assuntos
Purinérgicos/química , Purinérgicos/farmacologia , Purinérgicos/uso terapêutico , Receptores Purinérgicos P1/química , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2Y/química , Animais , Química Farmacêutica , Humanos , Agonistas do Receptor Purinérgico P1/química , Agonistas do Receptor Purinérgico P1/farmacologia , Agonistas do Receptor Purinérgico P1/uso terapêutico , Antagonistas de Receptores Purinérgicos P1/química , Antagonistas de Receptores Purinérgicos P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/uso terapêutico , Agonistas do Receptor Purinérgico P2Y/química , Agonistas do Receptor Purinérgico P2Y/farmacologia , Agonistas do Receptor Purinérgico P2Y/uso terapêutico , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Relação Estrutura-AtividadeRESUMO
Purinergic signaling in the nervous system has been the focus of a considerable number of studies since the 1970s. The P2X and P2Y receptors are involved in the initiation of purinergic signaling. They are very abundant in the central and peripheral nervous systems, where they are expressed on the surface of neurons and glial cells--microglia, astrocytes, oligodendrocytes and Schwann cells and the precursors of the latter two. Their ligands--extracellular nucleotides--are released in the physiological state by astrocytes and neurons forming synaptic connections, and are essential for the proper functioning of nervous system cells. Purinergic signaling plays a crucial role in neuromodulation, neurotransmission, myelination in the CNS and PNS, intercellular communication, the regulation of ramified microglia activity, the induction of the response to damaging agents, the modulation of synaptic activity and other glial cells by astrocytes, and the induction of astrogliosis. Understanding these mechanisms and the fact that P2 receptors and their ligands are involved in the pathogenesis of diseases of the nervous system may help in the design of drugs with different and more effective mechanisms of action.
Assuntos
Neurônios/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Trifosfato de Adenosina/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Cálcio/metabolismo , Humanos , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2Y/química , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Adenosine diphosphate ribose (ADPR), a product of ß-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. METHODS: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. RESULTS: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. CONCLUSION: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.
Assuntos
Adenosina Difosfato Ribose/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Estrenos/farmacologia , Imidazóis/farmacologia , Íons/química , Íons/metabolismo , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nifedipino/farmacologia , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y1/química , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suramina/farmacologia , Canais de Cátion TRPM/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Ligand-gated ion channels (LGICs) are cell surface integral proteins that mediate the fast neurotransmission in the nervous system. LGICs require auxiliary subunits for their trafficking, assembly and pharmacological modulation. Auxiliary subunits do not form functional homomeric receptors, but are reported to assemble with the principal subunits in order to modulate their pharmacological profiles. For example, nACh receptors are built at least by co-assemble of α and ß subunits, and the neuronal auxiliary subunits ß3 and α5 and muscle type ß, δ, γ, and ϵ determine the agonist affinity of these receptors. Serotonergic 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E are reported to assemble with the 5-HT3A subunit to modulate its pharmacological profile. Functional studies evaluating the role of γ2 and δ auxiliary subunits of GABAA receptors have made important advances in the understanding of the action of benzodiazepines, ethanol and neurosteroids. Glycine receptors are composed principally by α1-3 subunits and the auxiliary subunit ß determines their synaptic location and their pharmacological response to propofol and ethanol. NMDA receptors appear to be functional as heterotetrameric channels. So far, the existence of NMDA auxiliary subunits is controversial. On the other hand, Kainate receptors are modulated by NETO 1 and 2. AMPA receptors are modulated by TARPs, Shisa 9, CKAMP44, CNIH2-3 auxiliary proteins reported that controls their trafficking, conductance and gating of channels. P2X receptors are able to associate with auxiliary Pannexin-1 protein to modulate P2X7 receptors. Considering the pharmacological relevance of different LGICs auxiliary subunits in the present work we will highlight the therapeutic potential of these modulator proteins.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante/efeitos dos fármacos , Animais , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Modelos Moleculares , Subunidades Proteicas , Receptores de AMPA/química , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Receptores de Glicina/química , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/efeitos dos fármacos , Receptores de Ácido Caínico/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/efeitos dos fármacos , Receptores Purinérgicos P2X/metabolismo , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/metabolismoRESUMO
The P2X receptors are trimeric ATP-gated ion channels and mediate chemical communication between eukaryotic cells. Each P2X subunit contains two transmembrane helices, M1 and M2, and the M2 helix packs around an ion conduction pore. Here, I have reconstructed the three-dimensional models of the zebrafish P2X4 transmembrane domain using spatial restraints on helical packing. The models are stable in lipid bilayers during molecular dynamics simulation and adopt different conformations depending on bilayer hydrophobic thickness. Comparison of these conformations shows that the pore-lining residues L340, A344 and A347 each have multiple packing sites that define the pore configurations. Shift of L340 packing between different sites alters the side-chain orientation that occludes the pore or removes this occlusion. L340, A344 and A347 also gate the pore by expansion-contraction mechanism based on their packing patterns. Finally, pore expansions at the L340 and A344 levels are mutually exclusive, so the P2X gating may involve sequential pore opening at L340 and A344 levels to allow ion conduction. In summary, the current study shows that the computational assembly of the helical membrane protein is not only possible, but also necessary to provide insights into the mechanisms of channel gating.
Assuntos
Trifosfato de Adenosina/química , Ativação do Canal Iônico , Bicamadas Lipídicas/química , Simulação de Acoplamento Molecular , Receptores Purinérgicos P2X/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fosfatidilcolinas/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica , Peixe-ZebraRESUMO
ATP is a key energetic molecule, fundamental to cell function, which also has an important role in the extracellular milieu as a signaling molecule, acting as a chemoattractant for immune cells and as a neuro- and gliotransmitter. The ionotropic P2X receptors are members of an ATP-gated ion channels family. These ionotropic receptors are widely expressed through the body, with 7 subunits described in mammals, which are arranged in a trimeric configuration with a central pore permeable mainly to Ca(2+) and Na(+). All 7 subunits are expressed in different brain areas, being present in neurons and glia. ATP, through these ionotropic receptors, can act as a neuromodulator, facilitating the Ca(2+)-dependent release of neurotransmitters, inducing the cross-inhibition between P2XR and GABA receptors, and exercising by this way a modulation of synaptic plasticity. Growing evidence shows that P2XR play an important role in neuronal disorders and neurodegenerative diseases, like Parkinson's and Alzheimer's disease; this role involves changes on P2XR expression levels, activation of key pathways like GSK3ß, APP processing, oxidative stress and inflammatory response. This review is focused on the neuromodulatory function of P2XR on pathophysiological conditions of the brain; the recent evidence could open a window to a new therapeutic target.