- Publikační typ
- abstrakt z konference MeSH
The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATPγS and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions.
- MeSH
- adenosintrifosfát farmakologie MeSH
- antagonisté excitačních aminokyselin farmakologie MeSH
- biofyzikální jevy účinky léků MeSH
- blokátory sodíkových kanálů farmakologie MeSH
- GABA farmakologie MeSH
- inhibitory agregace trombocytů farmakologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- messenger RNA metabolismus MeSH
- metoda terčíkového zámku MeSH
- nervový přenos účinky léků MeSH
- nervový útlum účinky léků MeSH
- neurony účinky léků MeSH
- novorozená zvířata MeSH
- nucleus suprachiasmaticus cytologie MeSH
- potkani Wistar MeSH
- purinergní látky farmakologie MeSH
- purinergní receptory P2X genetika metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- synaptické potenciály účinky léků MeSH
- techniky in vitro MeSH
- tetrodotoxin farmakologie MeSH
- vápník metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.
- MeSH
- adenosintrifosfát chemie metabolismus MeSH
- alosterická regulace MeSH
- aminokyseliny chemie genetika metabolismus MeSH
- bodová mutace MeSH
- gating iontového kanálu účinky léků fyziologie MeSH
- HEK293 buňky MeSH
- hydrofobní a hydrofilní interakce MeSH
- ivermektin chemie farmakologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- metoda terčíkového zámku MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- purinergní receptory P2X4 chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- transfekce MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- Publikační typ
- abstrakt z konference MeSH
The supraoptic nuclei (SON), the hypothalamic release site of vasopressin and oxytocin, receive a non-glutamatergic, excitatory input from the caudal medulla that uses noradrenaline and ATP as neurotransmitters. Here, we studied the actions of extracellular ATP on SON neurons in hypothalamic slices isolated from the brains of 16- to 24-day-old rats. Whole-cell current clamp recordings performed 1-6 h after isolation showed that exogenous ATP application increased the frequency of action potentials and induced the depolarization of resting membranes. Voltage clamp recordings showed that ATP increased the frequency of GABAergic or glutamatergic spontaneous synaptic currents without changing their amplitude and evoked inward current (126±13 pA) in about 80% of SON neurons. The application of ATPγS and 2MeSATP mimicked the effects of ATP, but 2MeSADP, 2MeSAMP and αβmeATP had no effect. The P2X7 receptor agonist, BzATP, did not induce an inward current, but it increased intracellular calcium concentration in non-neuronal SON cells in slices. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) inhibited ATP-induced currents, whereas pH 6.5 and ivermectin, a specific allosteric modulator of the P2X4 receptor, potentiated ATP-induced currents. The P2Y1-selective antagonist, 2'-deoxy-N⁶-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), had no effect on ATP-induced responses. Quantitative real-time PCR showed that P2X2>P2X7>P2X4 purinergic receptor mRNAs were expressed in the SON tissue, but the levels of P2X1, P2X3, P2X5, P2X6, P2Y1, P2Y2 and P2Y12 mRNA were minor. These results show that SON neurons express functional presynaptic and extrasynaptic P2X2 and P2X4 receptors that modulate glutamate and GABA release and control the electrical excitability of SON neurons.
- MeSH
- adenosintrifosfát farmakologie MeSH
- akční potenciály účinky léků fyziologie MeSH
- GABA sekrece MeSH
- krysa rodu rattus MeSH
- kyselina glutamová sekrece MeSH
- metoda terčíkového zámku MeSH
- neurony účinky léků sekrece MeSH
- nucleus supraopticus účinky léků metabolismus MeSH
- orgánové kultury - kultivační techniky MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- potkani Wistar MeSH
- purinergní receptory P2X2 metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.
- MeSH
- cystein genetika chemie MeSH
- financování organizované MeSH
- gating iontového kanálu fyziologie MeSH
- HEK293 buňky MeSH
- konzervovaná sekvence MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- purinergní receptory P2X4 genetika chemie metabolismus MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata 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
- adenosintrifosfát analogy a deriváty farmakologie MeSH
- aminokyseliny aromatické genetika metabolismus MeSH
- biofyzika MeSH
- elektrická stimulace MeSH
- financování organizované MeSH
- konformace proteinů MeSH
- lidé MeSH
- membránové potenciály genetika účinky léků MeSH
- metoda terčíkového zámku metody MeSH
- mutageneze genetika MeSH
- purinergní receptory P2 genetika klasifikace metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce fyziologie účinky léků MeSH
- terciární struktura proteinů genetika fyziologie MeSH
- transfekce metody MeSH
- transformované buněčné linie MeSH
- vazba proteinů fyziologie genetika MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zelené fluorescenční proteiny MeSH
- Check Tag
- lidé 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
- adenosintrifosfát metabolismus MeSH
- gonadotropní buňky metabolismus MeSH
- hypofýza metabolismus MeSH
- hypothalamus metabolismus MeSH
- ivermektin chemie farmakologie MeSH
- kvarterní struktura proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- neuroglie metabolismus MeSH
- neurony metabolismus MeSH
- purinergní receptory P2 chemie účinky léků metabolismus MeSH
- sekundární struktura proteinů MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
