Purinergic P2X receptors (P2X) are ATP-gated ion channels that are broadly expressed in the brain, particularly in the hypothalamus. As ionic channels with high permeability to calcium, P2X play an important and active role in neural functions. The hypothalamus contains a number of small nuclei with many molecularly defined types of peptidergic neurons that affect a wide range of physiological functions, including water balance, blood pressure, metabolism, food intake, circadian rhythm, childbirth and breastfeeding, growth, stress, body temperature, and multiple behaviors. P2X are expressed in hypothalamic neurons, astrocytes, tanycytes, and microvessels. This review focuses on cell-type specific expression of P2X in the most important hypothalamic nuclei, such as the supraoptic nucleus (SON), paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), anteroventral periventricular nucleus (AVPV), anterior hypothalamic nucleus (AHN), arcuate nucleus (ARC), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), tuberomammillary nucleus (TMN), and lateral hypothalamic area (LHA).> The review also notes the possible role of P2X and extracellular ATP in specific hypothalamic functions. The literature summarized here shows that purinergic signaling is involved in the control of the hypothalamic-pituitary endocrine system, the hypothalamic-neurohypophysial system, the circadian systems and nonendocrine hypothalamic functions.

• Keywords
• P2X, arcuate nucleus (ARC), extracellular ATP, hypothalamus, paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), supraoptic nucleus (SON),
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Astrocytes metabolism   MeSH
• Hypothalamus * metabolism   cytology   MeSH
• Humans MeSH
• Neurons metabolism   MeSH
• Receptors, Purinergic P2X * metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Receptors, Purinergic P2X * MeSH

Allosteric modulators of ligand-gated receptor channels induce conformational changes of the entire protein that alter potencies and efficacies for orthosteric ligands, expressed as the half maximal effective concentration (EC50) and maximum current amplitude, respectively. Here, we studied the influence of allostery on channel pore dilation, an issue not previously addressed. Experiments were done using the rat P2X4 receptor expressed in human embryonic kidney 293T cells and gated by adenosine 5'-triphosphate (ATP) in the presence and absence of ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically induced sensitization of P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.

• MeSH
• Allosteric Regulation MeSH
• Ion Channel Gating * MeSH
• HEK293 Cells MeSH
• Ivermectin chemistry   pharmacology   MeSH
• Kinetics MeSH
• Rats MeSH
• Humans MeSH
• Receptors, Purinergic P2X4 chemistry   genetics   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding 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
• Names of Substances
• Ivermectin MeSH
• Receptors, Purinergic P2X4 MeSH

Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Purinergic P2X Receptor Agonists pharmacology   MeSH
• Alanine genetics   metabolism   MeSH
• Ion Channel Gating drug effects   MeSH
• HEK293 Cells MeSH
• Ivermectin pharmacology   MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Molecular Sequence Data MeSH
• Mutagenesis MeSH
• Receptors, Purinergic P2X4 chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Molecular Dynamics Simulation MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding 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
• Names of Substances
• Adenosine Triphosphate MeSH
• Purinergic P2X Receptor Agonists MeSH
• Alanine MeSH
• Ivermectin MeSH
• Receptors, Purinergic P2X4 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
• Protein Conformation MeSH
• Humans MeSH
• Membrane Potentials drug effects   genetics   MeSH
• Patch-Clamp Techniques methods   MeSH
• Mutagenesis genetics   MeSH
• Receptors, Purinergic P2 classification   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction drug effects   physiology   MeSH
• Protein Structure, Tertiary genetics   physiology   MeSH
• Transfection methods   MeSH
• Cell Line, Transformed MeSH
• Protein Binding genetics   physiology   MeSH
• Dose-Response Relationship, Drug MeSH
• Green Fluorescent Proteins genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• alpha,beta-methyleneadenosine 5'-triphosphate MeSH Browser
• Amino Acids, Aromatic MeSH
• enhanced green fluorescent protein MeSH Browser
• Receptors, Purinergic P2 MeSH
• Green Fluorescent Proteins MeSH