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
- Adenosine Triphosphate pharmacology MeSH
- Excitatory Amino Acid Antagonists pharmacology MeSH
- Biophysical Phenomena drug effects MeSH
- Sodium Channel Blockers pharmacology MeSH
- gamma-Aminobutyric Acid pharmacology MeSH
- Platelet Aggregation Inhibitors pharmacology MeSH
- Rats MeSH
- Cells, Cultured MeSH
- RNA, Messenger metabolism MeSH
- Patch-Clamp Techniques MeSH
- Synaptic Transmission drug effects MeSH
- Neural Inhibition drug effects MeSH
- Neurons drug effects MeSH
- Animals, Newborn MeSH
- Suprachiasmatic Nucleus cytology MeSH
- Rats, Wistar MeSH
- Purinergic Agents pharmacology MeSH
- Receptors, Purinergic P2X genetics metabolism MeSH
- Gene Expression Regulation drug effects MeSH
- Synaptic Potentials drug effects MeSH
- In Vitro Techniques MeSH
- Tetrodotoxin pharmacology MeSH
- Calcium metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Adenosine Triphosphate chemistry metabolism MeSH
- Allosteric Regulation MeSH
- Amino Acids chemistry genetics metabolism MeSH
- Point Mutation MeSH
- Ion Channel Gating drug effects physiology MeSH
- HEK293 Cells MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Ivermectin chemistry pharmacology MeSH
- Rats MeSH
- Humans MeSH
- Patch-Clamp Techniques MeSH
- Models, Molecular MeSH
- Mutagenesis, Site-Directed MeSH
- Receptors, Purinergic P2X4 chemistry genetics metabolism MeSH
- Protein Structure, Secondary MeSH
- Transfection MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Intramural 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
- Adenosine Triphosphate pharmacology MeSH
- Action Potentials drug effects physiology MeSH
- gamma-Aminobutyric Acid secretion MeSH
- Rats MeSH
- Glutamic Acid secretion MeSH
- Patch-Clamp Techniques MeSH
- Neurons drug effects secretion MeSH
- Supraoptic Nucleus drug effects metabolism MeSH
- Organ Culture Techniques MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Rats, Wistar MeSH
- Receptors, Purinergic P2X2 metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Cysteine genetics chemistry MeSH
- Financing, Organized MeSH
- Ion Channel Gating physiology MeSH
- HEK293 Cells MeSH
- Conserved Sequence MeSH
- Rats MeSH
- Humans MeSH
- Receptors, Purinergic P2X4 genetics chemistry metabolism MeSH
- Binding Sites MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals 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
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
