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1.
Bioinformatics ; 38(6): 1743-1744, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-34954796

RESUMO

SUMMARY: The 3D structure of transmembrane helices plays a key role in the function of membrane proteins. While visual inspection can usually discern the distinctive features of a helix bundle, simply translating them into a 2D diagram can be difficult. ATOLL (Aligned Transmembrane dOmains Layout fLattening) projects the helix bundle onto the lipid bilayer plane, thereby facilitating the comparison of different structures of the same membrane protein or structures of different membrane proteins. AVAILABILITY AND IMPLEMENTATION: ATOLL is a program written in Python3. The source code is freely available on the web at https://github.com/LIT-CCM-lab/ATOLL. ATOLL is implemented into a web server (https://atoll.drugdesign.unistra.fr/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Computadores , Software , Proteínas de Membrana
2.
Int J Mol Sci ; 24(13)2023 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-37446075

RESUMO

Macropore formation and current facilitation are intriguing phenomena associated with ATP-gated P2X7 receptors (P2X7). Macropores are large pores formed in the cell membrane that allow the passage of large molecules. The precise mechanisms underlying macropore formation remain poorly understood, but recent evidence suggests two alternative pathways: a direct entry through the P2X7 pore itself, and an indirect pathway triggered by P2X7 activation involving additional proteins, such as TMEM16F channel/scramblase. On the other hand, current facilitation refers to the progressive increase in current amplitude and activation kinetics observed with prolonged or repetitive exposure to ATP. Various mechanisms, including the activation of chloride channels and intrinsic properties of P2X7, have been proposed to explain this phenomenon. In this comprehensive review, we present an in-depth overview of P2X7 current facilitation and macropore formation, highlighting new findings and proposing mechanistic models that may offer fresh insights into these untangled processes.


Assuntos
Trifosfato de Adenosina , Receptores Purinérgicos P2X7 , Membrana Celular/metabolismo , Trifosfato de Adenosina/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207150

RESUMO

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.


Assuntos
Anoctaminas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Anoctaminas/química , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Colesterol/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Modelos Biológicos , Oócitos , Receptores Purinérgicos P2X7/química
4.
Proc Natl Acad Sci U S A ; 114(19): E3786-E3795, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28442564

RESUMO

Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as N-methyl-d-glucamine (NMDG+). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG+-permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG+ permeability superimposes that of Na+ and demonstrate that the molecular motions leading to the permeation of NMDG+ are very similar to those that drive Na+ flow. We found, however, that NMDG+ "percolates" 10 times slower than Na+ in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG+ but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.


Assuntos
Permeabilidade da Membrana Celular , Glutamatos/metabolismo , Modelos Biológicos , Receptores Purinérgicos P2X/metabolismo , Espermidina/metabolismo , Células HEK293 , Humanos
5.
Trends Biochem Sci ; 38(1): 20-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23206935

RESUMO

P2X receptors are nonselective cation channels gated by extracellular ATP. They represent new therapeutic targets, and they form channels with a unique trimeric architecture. In 2009, the first crystal structure of a P2X receptor was reported, in which the receptor was in an ATP-free, closed channel state. However, our view recently changed when a second crystal structure was reported, in which a P2X receptor was bound to ATP and resolved in an open channel conformation. This remarkable structure not only confirms many key experimental data, including the recent mechanisms of ATP binding and ion permeation, but also reveals unanticipated mechanisms. Certainly, this new information will accelerate our understanding of P2X receptor function and pharmacology at the atomic level.


Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2X/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores Purinérgicos P2X/química , Homologia de Sequência de Aminoácidos
6.
EMBO J ; 31(9): 2134-43, 2012 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-22473210

RESUMO

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


Assuntos
Trifosfato de Adenosina/fisiologia , Receptores Purinérgicos P2X2/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Zinco/farmacologia
7.
Proc Natl Acad Sci U S A ; 110(51): 20813-8, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24297890

RESUMO

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.


Assuntos
Compostos Azo/química , Ativação do Canal Iônico/efeitos da radiação , Luz , Neurônios/metabolismo , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Ratos
8.
Proc Natl Acad Sci U S A ; 109(28): 11396-401, 2012 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-22745172

RESUMO

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.


Assuntos
Cisteína/química , Receptores Purinérgicos P2X1/metabolismo , Animais , Cátions , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , DNA Complementar/metabolismo , Eletrofisiologia/métodos , Cinética , Maleimidas/química , Microscopia de Fluorescência/métodos , Mutação , Oócitos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Rodaminas/química , Xenopus/metabolismo , Xenopus laevis/metabolismo
9.
Proc Natl Acad Sci U S A ; 108(22): 9066-71, 2011 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-21576497

RESUMO

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.


Assuntos
Trifosfato de Adenosina/química , Receptores Purinérgicos P2X2/química , Sequência de Aminoácidos , Sítios de Ligação , Biofísica/métodos , Linhagem Celular , Membrana Celular/metabolismo , Cisteína/química , DNA Complementar/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos
10.
Nature ; 445(7123): 116-9, 2007 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17167423

RESUMO

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.


Assuntos
Cianobactérias/metabolismo , Ativação do Canal Iônico , Canais Iônicos/classificação , Canais Iônicos/metabolismo , Prótons , Receptores Nicotínicos/classificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Cianobactérias/genética , Condutividade Elétrica , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Células Procarióticas/metabolismo , Conformação Proteica , Receptores Nicotínicos/química , Xenopus
11.
Nat Commun ; 14(1): 1269, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36882406

RESUMO

PIEZO proteins are unusually large, mechanically-activated trimeric ion channels. The central pore features structural similarities with the pore of other trimeric ion channels, including purinergic P2X receptors, for which optical control of channel gating has been previously achieved with photoswitchable azobenzenes. Extension of these chemical optogenetics methods to mechanically-activated ion channels would provide tools for specific manipulation of pore activity alternative to non-specific mechanical stimulations. Here we report a light-gated mouse PIEZO1 channel, in which an azobenzene-based photoswitch covalently tethered to an engineered cysteine, Y2464C, localized at the extracellular apex of the transmembrane helix 38, rapidly triggers channel gating upon 365-nm-light irradiation. We provide evidence that this light-gated channel recapitulates mechanically-activated PIEZO1 functional properties, and show that light-induced molecular motions are similar to those evoked mechanically. These results push the limits of azobenzene-based methods to unusually large ion channels and provide a simple stimulation means to specifically interrogate PIEZO1 function.


Assuntos
Compostos Azo , Cisteína , Animais , Camundongos , Movimento (Física) , Optogenética , Canais Iônicos
12.
iScience ; 26(11): 108110, 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-37860691

RESUMO

In neuropathic pain, recent evidence has highlighted a sex-dependent role of the P2X4 receptor in spinal microglia in the development of tactile allodynia following nerve injury. Here, using internalization-defective P2X4mCherryIN knockin mice (P2X4KI), we demonstrate that increased cell surface expression of P2X4 induces hypersensitivity to mechanical stimulations and hyperexcitability in spinal cord neurons of both male and female naive mice. During neuropathy, both wild-type (WT) and P2X4KI mice of both sexes develop tactile allodynia accompanied by spinal neuron hyperexcitability. These responses are selectively associated with P2X4, as they are absent in global P2X4KO or myeloid-specific P2X4KO mice. We show that P2X4 is de novo expressed in reactive microglia in neuropathic WT and P2X4KI mice of both sexes and that tactile allodynia is relieved by pharmacological blockade of P2X4 or TrkB. These results show that the upregulation of P2X4 in microglia is crucial for neuropathic pain, regardless of sex.

13.
Methods Mol Biol ; 2510: 239-252, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35776328

RESUMO

P2X7 receptors are ATP-gated ion channels permeable to metal cations, such as Na+, K+, and Ca2+. They also exhibit permeability to various large molecular weight species, reaching up to 900 Da, in a process known as macropore formation, which is a unique functional hallmark across the P2X family. While well-documented in a range of different cell types, the molecular mechanism underlying this phenomenon is poorly understood, and has been clouded through the use of electrophysiological methodology prone to artifacts as a result of significant changes in ionic concentrations in asymmetric conditions. In this chapter, we discuss the permeation properties of P2X7, the related methodological challenges and the use of symmetrical organic cation solutions as a useful technique for probing P2X7 permeation.


Assuntos
Sódio , Cátions
14.
Curr Opin Pharmacol ; 62: 109-116, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34965483

RESUMO

Ligand-gated ion channels (LGIC, also referred to as ionotropic receptors) are important transmembrane proteins that open to allow ions to flow across the membrane and locally modify the membrane potential in response to the binding of a ligand. For more than a decade, a tremendous effort has been carried out in the determination of many LGIC structures in high resolution, leading to an unprecedented molecular description of channel gating. However, it is sometimes difficult to classify experimentally derived structures to their corresponding functional states, and alternative methods may help resolve or refine this issue. In this review, we focus on the application of photo-isomerizable tweezers (PIT) as a powerful strategy to interrogate molecular mechanisms of LGIC while assessing their functionality by electrophysiology.


Assuntos
Potenciais da Membrana , Humanos , Ligantes
15.
J Biol Chem ; 285(21): 15805-15, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20308075

RESUMO

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu(63) and Arg(274) produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu(63) and Arg(274) contributes to the ion channel function. Engineered cysteines E63C and R274C formed redox-dependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu(63) and Arg(274), allowing the transmembrane helices to open the channel.


Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Animais , Linhagem Celular , Dissulfetos/metabolismo , Humanos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2
16.
Methods Enzymol ; 653: 349-376, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34099179

RESUMO

The modulation of ion channel activity is of central importance within the nervous system, and an in-depth understanding of how such activity occurs on the molecular level is of prime importance for enhancing our understanding of neuronal systems in physiological and pathological states. The use of light as a stimulus has presented the unique opportunity to study these dynamic processes with exquisite spatiotemporal control. We have developed the photoswitchable tweezers method, an optogenetic pharmacology-based technique which relies on the use of a photoswitchable crosslinker as "tweezers" to manipulate the molecular movements involved in ion channel functionalities. Not only does this allow optical control of ion channel activity, but also investigation into the molecular motions and inter-residue distances implicated in such activity. In this chapter we discuss the principles behind the photoswitchable tweezers method, its strategic design and the key experimental steps involved in this technique, using purinergic P2X2 receptor as a case study system.


Assuntos
Ativação do Canal Iônico , Canais Iônicos , Trifosfato de Adenosina , Canais Iônicos/genética , Canais Iônicos/metabolismo , Neurônios/metabolismo , Optogenética
17.
eNeuro ; 8(1)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33380526

RESUMO

ATP is an extracellular signaling molecule involved in numerous physiological and pathologic processes. However, in situ characterization of the spatiotemporal dynamic of extracellular ATP is still challenging because of the lack of sensor with appropriate specificity, sensitivity, and kinetics. Here, we report the development of biosensors based on the fusion of cation permeable ATP receptors (P2X) to genetically encoded calcium sensors [genetically encoded calcium indicator (GECI)]. By combining the features of P2X receptors with the high signal-to-noise ratio of GECIs, we generated ultrasensitive green and red fluorescent sniffers that detect nanomolar ATP concentrations in situ and also enable the tracking of P2X receptor activity. We provide the proof of concept that these sensors can dynamically track ATP release evoked by depolarization in mouse neurons or by extracellular hypotonicity. Targeting these P2X-based biosensors to diverse cell types should advance our knowledge of extracellular ATP dynamics in vivo.


Assuntos
Receptores Purinérgicos P2 , Trifosfato de Adenosina , Animais , Cálcio , Camundongos , Neurônios , Receptores Purinérgicos P2/genética , Transdução de Sinais
18.
Br J Pharmacol ; 178(3): 489-514, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33125712

RESUMO

The known seven mammalian receptor subunits (P2X1-7) form cationic channels gated by ATP. Three subunits compose a receptor channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1 and TM2), intracellular N- and C-termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D structure and gating cycle of P2X receptors. The agonist-binding pocket is located at the intersection of two neighbouring subunits. In addition to the mammalian P2X receptors, their primitive ligand-gated counterparts with little structural similarity have also been cloned. Selective agonists for P2X receptor subtypes are not available, but medicinal chemistry supplied a range of subtype-selective antagonists, as well as positive and negative allosteric modulators. Knockout mice and selective antagonists helped to identify pathological functions due to defective P2X receptors, such as male infertility (P2X1), hearing loss (P2X2), pain/cough (P2X3), neuropathic pain (P2X4), inflammatory bone loss (P2X5), and faulty immune reactions (P2X7).


Assuntos
Trifosfato de Adenosina , Animais , Ligantes , Masculino , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2X2
20.
J Gen Physiol ; 151(7): 898-911, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126967

RESUMO

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.


Assuntos
Agonistas do Receptor Purinérgico P2X/metabolismo , Receptores Purinérgicos P2X2/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Agonistas do Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X2/metabolismo , Xenopus laevis
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