Purinergic P2X receptors (P2XR), activated by extracellular adenosine 5‘-triphosphate (ATP), represent a specific type of li- gand-gated ion channels. They form functional trimeric homomers or heteromers which are nonselectively cation-perme- able after receptor activation. P2X receptors are widely expressed in excitable and nonexcitable tissues and are involved in many physiological and pathophysiological processes such as platelet aggregation, contraction of smooth muscle, immu- ne responses, cell proliferation and apoptosis or neurotransmission. In mammals, seven P2X subunits (P2X1-P2X7) have been identified. They differ mainly in distribution, pharmacological profile and kinetics of ATP-induced responses. The sub- type P2X7 is the most specific in the P2X family and widely differs from other P2X subtypes.
- Keywords
- extracelulární ATP, purinergní P2X rodina, buněčná proliferace a apoptóza,
- MeSH
- Adenosine Triphosphate biosynthesis MeSH
- Purinergic P2X Receptor Agonists pharmacokinetics pharmacology MeSH
- Apoptosis physiology MeSH
- Extracellular Space MeSH
- Cell Physiological Phenomena MeSH
- Humans MeSH
- Intercellular Signaling Peptides and Proteins MeSH
- Cell Proliferation MeSH
- Receptors, Purinergic P2X * analysis biosynthesis physiology isolation & purification classification metabolism drug effects MeSH
- Receptors, Purinergic P2X1 blood MeSH
- Receptors, Purinergic P2X3 physiology MeSH
- Receptors, Purinergic P2X4 MeSH
- Receptors, Purinergic P2X7 immunology MeSH
- Signal Transduction MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Keywords
- extracelulární ATP,
- MeSH
- Adenosine Triphosphate physiology MeSH
- Chronic Pain etiology MeSH
- Extracellular Space MeSH
- Humans MeSH
- Microglia MeSH
- Synaptic Transmission * physiology MeSH
- Neuralgia * etiology MeSH
- Peripheral Nerve Injuries MeSH
- Receptors, Purinergic P2X * physiology MeSH
- Receptors, Purinergic P2X3 physiology MeSH
- Receptors, Purinergic P2X4 physiology MeSH
- Receptors, Purinergic P2X7 physiology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Neurosteroids are steroids synthesized de novo in the brain from cholesterol in an independent manner from peripheral steroid sources. The term "neuroactive steroid" includes all steroids independent of their origin, and newly synthesized analogs of neurosteroids that modify neuronal activities. In vivo application of neuroactive steroids induces potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the γ-aminobutyric acid type-A receptor (GABAAR). However, neuroactive steroids also act as positive or negative allosteric regulators on several ligand-gated channels including N-methyl-d-aspartate receptors (NMDARs), nicotinic acetylcholine receptors (nAChRs) and ATP-gated purinergic P2X receptors. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain and can be regulated by neurosteroids. Transmembrane domains are necessary for neurosteroid binding, however, no generic motif of amino acids can accurately predict the neurosteroid binding site for any of the ligand-gated ion channels including P2X. Here, we will review what is currently known about the modulation of rat and human P2X by neuroactive steroids and the possible structural determinants underlying neurosteroid-induced potentiation and inhibition of the P2X2 and P2X4 receptors. This article is part of the Special Issue on "Purinergic Signaling: 50 years".
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Ligand-Gated Ion Channels * metabolism MeSH
- Rats MeSH
- Humans MeSH
- Brain metabolism MeSH
- Neurosteroids * MeSH
- Receptors, Purinergic P2X metabolism MeSH
- Receptors, Purinergic P2X2 metabolism MeSH
- Binding Sites MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.
- MeSH
- Pituitary Gland, Anterior cytology drug effects physiology MeSH
- Purinergic P2X Receptor Agonists pharmacology MeSH
- Purinergic P2X Receptor Antagonists pharmacology MeSH
- Ion Channel Gating drug effects MeSH
- HEK293 Cells MeSH
- Hypothalamus cytology drug effects physiology MeSH
- Lithocholic Acid analogs & derivatives pharmacology MeSH
- Humans MeSH
- Neurons drug effects physiology MeSH
- Rats, Wistar MeSH
- Receptors, Purinergic P2X2 physiology MeSH
- Receptors, Purinergic P2X4 physiology MeSH
- Receptors, Purinergic P2X7 physiology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X(4) receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X(4)-TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln(36), Leu(40), Val(43), Val(47), Trp(50), Asn(338), Gly(342), Leu(346), Ala(349), and Ile(356) mutants. The substitution-sensitive Arg(33) and Cys(353) mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met(31), Tyr(42), Gly(45), Val(49), Gly(340), Leu(343), Ala(344), Gly(347), Thr(350), Asp(354), and Val(357) map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.
- MeSH
- Antiparasitic Agents metabolism MeSH
- Cell Line MeSH
- Financing, Organized MeSH
- Ion Channel Gating MeSH
- Ivermectin metabolism MeSH
- Protein Conformation MeSH
- Rats MeSH
- Humans MeSH
- Patch-Clamp Techniques MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Mutation MeSH
- Receptors, Purinergic P2 genetics chemistry metabolism MeSH
- Receptors, Purinergic P2X4 MeSH
- Amino Acid Sequence MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
Purinergic P2X receptors represent a novel structural type of ligand-gated ion channels activated by extracellular ATP. So far, seven P2X receptor subunits have been found in excitable as well as non-excitable tissues. Little is known about their structure, mechanism of channel opening, localization, and role in the central nervous system. The aim of this work is to summarize recent investigations and describe our contribution to elucidating the structure of the ATP binding site and transmembrane domains of the P2X receptor, we also discuss the expression and physiological roles played by the ATP and P2X receptors in the anterior pituitary and hypothalamus.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Gonadotrophs metabolism MeSH
- Pituitary Gland metabolism MeSH
- Hypothalamus metabolism MeSH
- Ivermectin chemistry pharmacology MeSH
- Protein Structure, Quaternary MeSH
- Humans MeSH
- Models, Molecular MeSH
- Neuroglia metabolism MeSH
- Neurons metabolism MeSH
- Receptors, Purinergic P2 chemistry drug effects metabolism MeSH
- Protein Structure, Secondary MeSH
- Protein Structure, Tertiary MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and alphabeta-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.
- MeSH
- Adenosine Triphosphate analogs & derivatives pharmacology MeSH
- Amino Acids, Aromatic genetics metabolism MeSH
- Biophysics MeSH
- Electric Stimulation MeSH
- Financing, Organized MeSH
- Protein Conformation MeSH
- Humans MeSH
- Membrane Potentials genetics drug effects MeSH
- Patch-Clamp Techniques methods MeSH
- Mutagenesis genetics MeSH
- Receptors, Purinergic P2 genetics classification metabolism MeSH
- Amino Acid Sequence MeSH
- Signal Transduction physiology drug effects MeSH
- Protein Structure, Tertiary genetics physiology MeSH
- Transfection methods MeSH
- Cell Line, Transformed MeSH
- Protein Binding physiology genetics MeSH
- Dose-Response Relationship, Drug MeSH
- Green Fluorescent Proteins MeSH
- Check Tag
- Humans MeSH
