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101.
Yuichi Minato Shiho Suzuki Tomoaki Hara Yutaka Kofuku Go Kasuya Yuichiro Fujiwara Shunsuke Igarashi Ei-ichiro Suzuki Osamu Nureki Motoyuki Hattori Takumi Ueda Ichio Shimada 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(17):4741-4746
Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s−1), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.In chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channels gated by extracellular ATP (1, 7–9) and are involved in many physiological and pathophysiological processes (10–12). Seven subtypes of the P2X receptors have been identified in mammals (13), and they share ∼40% sequence identity. The P2X4 receptor is involved in the pathogenesis of chronic neuropathic, inflammatory pain and the endothelial cell-mediated control of vascular tone (11, 14, 15). Compared with ATP, α,β-methylene ATP (α,β-meATP), in which the oxygen atom linking the α- and β-phosphorous atoms of ATP is replaced by a methylene group (Fig. S1A), reportedly induces a lower maximum current in cells expressing the mouse, rat, and human P2X4 receptors and other P2X receptors (16, 17).Open in a separate windowFig. S1.Characterization of the P2X4 receptor. (A) Chemical structures of ATP and α,β-meATP. (B and C) TEVC recordings of ATP- and α,β-meATP-evoked currents from rat P2X4 receptor expressed in Xenopus oocytes, respectively. In B, the currents were evoked twice by ATP (30 μM, 1 min, black bar). In C, the currents were firstly evoked by ATP (30 μM, 1 min, black bar) and subsequently by α,β-meATP (300 μM, 1 min, black bar). (D) TEVC recording of the ATP-evoked current (30 μM, 30 s, black bar) from the N-terminally EGFP-tagged ΔzfP2X4–A′ construct expressed in Xenopus oocytes. (E) Size exclusion chromatogram of purified EGFP-tagged ΔzfP2X4–A′ in rHDLs. Elution volumes corresponding to 17.0, 12.2, 10.4, and 7.1 nm Stokes diameters were determined by thyroglobulin, ferritin, catalase, and BSA, respectively. V0 and 1CV are void volume and single column volume, respectively. (F) SDS/PAGE analyses of purified ΔzfP2X4–A′ embedded in rHDLs. The samples were analyzed by 12% SDS/PAGE with Coomassie Brilliant Blue staining. (G) Measurement of [3H]ATP saturation binding to the purified ΔzfP2X4–A′ in rHDLs. (H and I) Estimation of the effects of deuteration based on the crystal structures of zfP2X4 (PDB ID code 4DW1) and the deuteration incorporation rates. The plots on the Left (without deuteration) and the Right (with deuteration) are the sums of the inverse sixth power of the distances between pseudoatoms centered on the methyl hydrogens of M108, M249, M268, or M325 and each hydrogen atom in the crystal structure of zfP2X4 (sums of the r−6) and the sums of the r−6 multiplied by [1 − (deuterium incorporation rates)] of each hydrogen atom, respectively. The graphs in H and I were calculated from the crystal structure in the apo state (PDB ID code 4DW0) and that in the ATP-bound state (PDB ID code 4DW1), respectively. Sums of the r−6 of each methionine methyl group and Hαβγ of the intraresidue methionine (green), Hαβγ of the interresidue methionine (light green), Hαβ of tyrosine (light violet), Hδεζη of tryptophan (orange), Hαβδεζ of phenylalanine (pink), Hαβγ of valine (blue), Hαβγδ of leucine (light blue), Hαβγδ of isoleucine (cyan), Hαβγ of threonine (light cyan), Hαβ of alanine (red), Hαβγδ of arginine (dark blue), Hα of glycine (dark green), and Hαβ of serine (magenta) residues, and the other hydrogens connected to carbon atoms (other unexchangeable hydrogens, light gray) are shown with colors. Hydrogen atoms connected to nitrogen, oxygen, or sulfur atoms were not considered in these calculations because these hydrogens should be exchanged with deuterium in D2O. The deuterium incorporation rates of the hydrogen atoms within each methionine residue (intraresidue) and the deuterium incorporation rates of other methionine residues (interresidue) were set to 98% and 85%, respectively, because the methionine residues would be derived from 85% of [α-, β-, γ-98% 2H-, methyl-13C]-methionine and 15% of nonlabeled methionine in the medium.The crystal structures of zebrafish P2X4 receptor (zfP2X4) (18, 19), together with mutational analyses (20–26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X4 forms a homotrimer (27, 28), in which the transmembrane region of each subunit is composed of two helices (19). In the crystal structure of zfP2X4 in the ATP-bound state, three ATP molecules are bound to the intersubunit nucleotide binding pockets. In addition, the region that connects the ATP-binding site and the transmembrane region, which is referred to as the “lower body” (Fig. 1 A and B), is expanded by ∼10 Å in the ATP-bound state, and a pore is formed in the transmembrane region, which is proposed to expand by the iris-like movement of the transmembrane helices (18). However, the mechanism underlying the partial activation of P2X receptors is unknown because the structures of the P2X receptors have not been examined in the partial agonist-bound states.Open in a separate windowFig. 1.NMR resonances from the endogenous methionine residues of zfP2X4 in rHDL. (A and B) Distribution of the methionine residues in the ΔzfP2X4–A′. One subunit from the crystal structure of zfP2X4 in the apo form (A) (PDB ID code 4DW0) and one from the ATP-bound form (B) (PDB ID code 4DW1) are shown in ribbons. The lower body and the right flipper are yellow. The A330 residues, the methionine residues, and the residues in which methionine mutations were introduced, L339 and L351, are depicted by green sticks. ATP is depicted by red sticks. Dummy atoms generated by Orientations of Proteins in Membranes (OPM), which represent membrane boundary planes, are gray. (C) Overlaid 1H-13C HMQC spectra of [2H-11AA, α, β-2H, methyl-13C-Met]ΔzfP2X4-A′, embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). The regions with resonances from methionine residues are shown, and the assigned resonances are indicated. The centers of the resonances are indicated with dots. Cross-sections at lines through the centers of each resonance in the ATP-bound state and the cross-sections of the spectra using [α, β-2H, methyl-13C-Met]ΔzfP2X4-A′ are shown on the top of the overlaid spectra. The intensities of the cross-sections were normalized by the concentration of ΔzfP2X4-A′ and the conditions of the NMR measurements.The P2X4 receptor used in the previous crystallographic studies was solubilized by detergents, which are widely used for structural investigations of membrane proteins, but the P2X4 receptor is embedded in lipid bilayers under physiological conditions. It was recently reported that reconstituted high-density lipoproteins (rHDLs), which are also known as nanodiscs (29), can accommodate membrane proteins within a 10-nm-diameter disk-shaped lipid bilayer (30). The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32–34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.Here, we used NMR to observe the conformational equilibrium of the alanine and methionine residues of zfP2X4 bound to α,β-meATP in rHDLs. Based on the conformational equilibrium, we discuss the mechanism underlying the partial activation of P2X receptors. 相似文献
102.
The mechanism by which intestinal secretagogues evoke fluid secretion in the small bowel and colon has been suggested to involve mucosal adenylate cyclase. Adenylate cyclase activity was assayed by conversion of [32P]ATP to [32P]cyclic AMP in a system of pure epithelial cells isolated from the small intestine of the hamster by vibration in buffer. Several gastrointestinal hormones were tested for their capacity to stimulate adenylate cyclase; vasoactive intestinal peptide and impure cholecystokinin-pancreozymin (but not the 99% pure preparation or pure cholecystokinin octapeptide) were potent stimuli, but pentagastrin, glucagon, secretin, and gastric inhibitory peptide were impotent. Two prostaglandins, PGE1 and PGE2, were potent stimuli of adenylate cyclase. Two other compounds that provoke intestinal secretion of fluid, deoxycholic acid and ricinoleic acid (castor oil), were ineffective stimuli of adenylate cyclase. These experiments do not support a clear-cut relationship between a compound's ability to stimulate adenylate cylase and its activity as an intestinal secretagogue. 相似文献
103.
The aim of presented study was analysis of fungal invasions of the genital organs, oral cavity and digestive tract in 799 women. Fungal strains were identified using API 20C and API 20C AUX (bioMérieux); they were as follows: Candida albicans, C. krusei, C. tropicalis, C. guilliermondii, C. kefyr, C. glabrata, C. famata, C. parapsilosis, Rhodotorula rubra, Geotrichum candidum. The number of fungal strains isolated from the oral cavity ontocenosis was the highest (41.8 +/- 2.4%). Fungal strains isolated from the vagina were less numerous (34.6 +/- 2.3%), and from the rectum the least numerous (23.6 +/- 2.1%). Multifocal invasions (limited to one ontocenosis) were present in about 50% of women; bifocal were less frequent, and this difference was statistically significant (p < 0.01): fungi in the vagina and oral cavity were detected in 28.6 +/- 3.0%, in the oral cavity and rectum in 19.0 +/- 2.6%, and in the vagina and rectum in 13.0 +/- 2.2%. Trifocal invasions were more frequent (39.4 +/- 3.2%). There was a visible trend of increasing prevalence of fungi in genital organs over 10 year period from 17.9 +/- 1.4% to 34.5 +/- 1.7%. 相似文献
104.
Shuying Jiang Makoto Naito Chikako Kaizu Kazuhisa Kuwata Go Hasegawa Naofumi Mukaida Leonard D. Shultz 《Liver international》2000,20(6):465-474
Abstract: Background/Aims: Mice homozygous for the osteopetrosis (op) mutation are genetically deficient in macrophage colony‐stimulating factor (M‐CSF/CSF‐1) and are characterized by defective differentiation and function of macrophages. The aim of this study is to assess the contribution of M‐CSF to lipopolysaccharide (LPS)‐induced cytokine expression and neutrophil infiltration in the liver. Methods: We investigated the effects of LPS administration in M‐CSF‐deficient op/op mutant mice. The expression of cytokines and receptors in the liver was studied by immunohistochemistry and RT‐PCR. Neutrophil infiltration in the liver was also examined. Results: After LPS administration, cytokine production and expression of LPS receptors, such as CD14 and scavenger receptor class A (MSR‐A), were induced at lower levels in op/op mice than those in littermate mice. Neutrophil infiltration in the liver of op/op mice did not differ significantly from that of littermate mice. Anti‐IL‐8 receptor homologue and anti‐C5a receptor antibody reduced the number of infiltrating neutrophils. Conclusions: These findings indicate that deficient macrophage activation following LPS injection in op/op mice is associated with decreased expression of CD14 and MSR‐A in the liver. Thus, M‐CSF plays a critical role in LPS‐induced macrophage activation but does not exert a dominant role in neutrophil infiltration in the liver. 相似文献
105.
Aging is associated with decreases in food intake and GH secretion, termed the anorexia of aging and somatopause, respectively. The mechanisms underlying these phenomena are not fully understood. Although many approaches have attempted to improve these age-related physiological changes, none have achieved satisfactory results. Ghrelin, a 28-amino-acid acylated peptide, was identified as an endogenous ligand for the GH secretagogue receptor. Ghrelin stimulates GH secretion and food intake in animals and humans. Previous studies have demonstrated that the mean plasma concentrations of ghrelin in normal-weight elderly people were lower than those in younger people. We hypothesized that ghrelin administration might improve the metabolic and physiological changes that accompany the anorexia of aging and somatopause. First, 75-wk-old mice fasted for 72 h, after which they resumed feeding with sc administration of ghrelin (360 microg/kg) twice daily for 4 d. Multiple administrations of ghrelin after a 72-h fast increased food intake and hastened body weight recovery with a high lean body mass ratio. Next, 50-wk-old mice were sc injected with rat ghrelin (40 microg/kg) twice weekly from 50-80 wk of age. Long-term administration of ghrelin kept aged mice with low body weight and low adiposity. These results suggest that ghrelin might be a novel approach for the therapy of age-related metabolic and physiological changes. 相似文献
106.
Kazutoshi Higuchi Seiji Futagami Hiroshi Yamawaki Makoto Murakami Kumiko Kirita Shuhei Agawa Go Ikeda Hiroto Noda Yasuhiro Kodaka Nobue Ueki Keiko Kaneko Katya Gudis Ryuji Ohashi Katsuhiko Iwakiri 《Journal of Clinical Biochemistry and Nutrition》2021,68(1):86
Since the prevention of early chronic pancreatitis (ECP) into chronic pancreatitis might be critical for the reduction of pancreatic cancer, we tried to clarify the pathophysiology of ECP patients, focusing on ECP patients without alcoholic chronic pancreatitis. 27 ECP patients without alcoholic chronic pancreatitis and 33 patients with functional dyspepsia with pancreatic enzyme abnormalities (FD-P) were enrolled in this study. Diagnosis of ECP was made when imaging findings showed the presence of more than 2 out of 7 endoscopic ultrasound features. Duodenal degranulated eosinophils and glucagon-like peptide 1 producing cells were estimated by immunostaining. There were no significant differences in characteristics and psychogenic factors between ECP and FD-P patients. Interestingly, endoscopic ultrasound score in ECP patients significantly improved, albeit clinical symptoms in ECP patients showed no improvement at one year follow up. The extent of migration of duodenal degranulated eosinophils in FD-P patients was significantly higher compared to that in ECP patients. The levels of elastase-1 and trypsin in ECP patients with improved endoscopic ultrasound features were significantly reduced by the treatment. Further studies will be needed to clarify whether clinical symptoms and endoscopic ultrasound features in ECP patients without alcoholic chronic pancreatitis were improved in longer follow up study. 相似文献
107.
108.
Ogura Sho Kimura Muneyoshi Takagi Shinsuke Mitsuki Takashi Yuasa Mitsuhiro Kageyama Kosei Kaji Daisuke Nishida Aya Taya Yuki Ishiwata Kazuya Yamamoto Hisashi Asano-Mori Yuki Yamamoto Go Uchida Naoyuki Wake Atsushi Taniguchi Shuichi Araoka Hideki 《European journal of clinical microbiology & infectious diseases》2021,40(5):941-948
European Journal of Clinical Microbiology & Infectious Diseases - The aim of this study is to clarify the characteristics of gram-negative bacteremia (GNB), including extended-spectrum... 相似文献
109.
Sekikawa Yuya Funada Keita Akamatsu Go Himuro Kazuhiko Takahashi Akihiko Baba Shingo Sasaki Masayuki 《Annals of nuclear medicine》2021,35(7):823-833
Annals of Nuclear Medicine - To examine the impact of acquisition time on Lutetium-177 (177Lu) single-photon emission computed tomography (SPECT) images using Monte Carlo simulation. A gamma camera... 相似文献
110.