Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

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.     

Impact of reflux esophagitis on the esophageal function before and after laparoscopic fundoplication

Background

High-resolution manometry (HRM), which is breakthrough testing equipment to evaluate esophageal motor function, was developed in Europe and United State and has garnered attention. Moreover, multichannel intraluminal impedance pH (MII-pH) testing has allowed us to grasp all liquid/gas reflux including not only acid but also non-acid reflux. We examined the impact of the presence of reflux esophagitis (RE) on esophageal motor function before and after laparoscopic fundoplication.

Materials and methods

The subjects included 100 patients (male: 63 patients, mean age: 54.1?±?15.8) among 145 patients who underwent laparoscopic fundoplication for GERD associated diseases during a 4-year period from October 2012 to September 2016, excluding 6 patients who underwent further surgery, 32 patients on whom HRM was not performed, 3 patients who had technical errors during testing, and 4 patients for whom the status of RE was unknown. Regarding HRM, Mano Scan from Given Imaging Ltd. was used, and for the analysis, Mano View version 3.0 from the same company was used, after which data was calculated based on the Chicago Classification advocated by Pandolfino et al. Moreover, for the MII-pH testing, Sleuth manufactured by Sandhill Scientific. Inc. was used and automatic analysis was conducted by a computer. Postoperative assessments were conducted 3 months following surgery for all. Data was described in the median value and inter-quartile range, with a statistically significant difference defined as p?<?0.05 by Chi square, Mann–Whitney, and Wilcoxon tests.

Results

RE+?group (Los Angeles classification A:B:C:D?=?7:9:16:12 patients) included 44 patients (44%), of older age compared to the RE? group (62 vs. 50 years, p?=?0.012) and a higher Body Mass Index value (24.0 vs. 22.5, p?=?0.045); however, no differences were observed in terms of gender and duration of symptoms. In the preoperative findings on MII-pH, the RE+?group demonstrated significantly longer acid reflux time (4.7 vs. 1.3%, p?=?0.005), while in the HRM findings, the RE? group demonstrated a significantly longer abdominal esophagus (0 vs. 0.4 cm, p?=?0.049) and maintained esophageal body motor function (DCI: 1054 vs. 1407 mmHg s cm, p?=?0.021, Intact peristalsis ratio: 90 vs. 100%, p?=?0.037). As to the comparison of the treatment effect before and after laparoscopic fundoplication (Toupet fundoplication for all), significant improvements were observed in both groups in various parameters regarding reflux including acid reflux time, total number of liquid reflux episodes and total number of reflux episodes. Moreover, for both groups, the total length of the lower esophageal sphincter (LES) (RE+?group: 2.7 vs. 3.2 cm, p?=?0.001, RE? group: 3.0 vs. 3.4 cm, p?=?0.003) and the total length of the abdominal esophagus (RE+?group: 0 vs. 1.6 cm, p?<?0.001, RE? group: 0 vs. 1.8 cm, p?=?0.001) were significantly extended following surgery; however, no change was observed in DCI before and after surgery.

Conclusions

Regardless of the presence of RE, cardiac function and LES function were improved following laparoscopic Toupet fundoplication, but no changes were observed in esophageal body motor function.

     

Accumulation of copper in the liver and hepatic injury in chronic hepatitis C

BACKGROUND AND AIMS: Relationships between chronic liver disease and trace metals have not been clearly understood. To examine connections between severity of hepatic fibrosis in chronic hepatitis C and copper, iron and zinc we measured the contents of these metals in liver tissue and serum in the patients. METHODS: Forty-one patients (26-62 years), 13 with fibrosis representing grade F1, 16 with F2, seven with F3, and five with F4, entered this study. Metals were quantified in needle liver-biopsy specimens by particle-induced X-ray emission. In serum, metals were measured by flameless atomic absorption spectrometry. RESULTS: Hepatic copper content increased with progression of hepatic fibrosis (P < 0.05). The copper content correlated positively with bilirubin (r = 0.466, P = 0.0023), and with type IV collagen (r = 0.402, P = 0.0086) and correlated negatively with albumin (r = -0.404, P = 0.080). However, hepatic iron and zinc contents did not show a significant differences between grades of fibrosis. CONCLUSIONS: Copper accumulation in fibrotic livers caused by chronic hepatitis C may contribute to hepatic injury. The real mechanism is not known at present, but excess copper may damage the liver by oxidative stress.     

A collision tumor of solitary fibrous tumor/hemangiopericytoma and meningioma: A case report with literature review

An intracranial collision tumor is a rare lesion composed of two histologically different neoplasms in the same anatomic location. Even more rare is the collision tumor of a solitary fibrous tumor/hemangiopericytoma (SFT/HPC) and meningioma. The patient was a 46-year-old woman who had a 40 × 35 × 30-mm mass in the vermis of the cerebellum. Histologically, the mass consisted of two different components. One component showed the morphology of meningioma (World Health Organization (WHO) grade I), and the other component exhibited small round cell proliferation with hypercellular density, which was revealed to be SFT/HPC (WHO grade III) based on STAT6 immunohistochemistry. STAT6 showed completely different immunohistochemistry results in these two components (nuclear-negative in meningioma and nuclear-positive in SFT/HPC). Since these two neoplasms are associated with different prognoses, they should be distinguished from each other. When meningioma and an SFT/HPC-like lesion are identified morphologically, it is important to recognize the presence of such a collision tumor composed of meningioma and SFT/HPC, and identify the SFT/HPC component by employing STAT6 immunohistochemistry.     

First experience of efficacy and radiation exposure in 320-detector row CT fluoroscopy-guided interventions

Objective:We investigated the efficacy and exposure to radiation in 320-detector row computed tomography fluoroscopy-guided (CTF-guided) interventions.Methods:We analysed 231 320-detector row CTF-guided interventions (207 patients over 2 years and 6 months) in terms of technical success rates, clinical success rates, complications, scanner settings, overall radiation doses (dose–length product, mGy*cm), patient doses of peri-interventional CT series, and interventional CT (including CTF), as a retrospective cohort study. The relationships between patient radiation dose and interventional factors were assessed using multivariate analysis.Results:Overall technical success rate was 98.7% (228/231). The technical success rates of biopsies, drainages, and aspirations were 98.7% (154/156), 98.5% (66/67), and 100% (8/8), respectively. The clinical success rate of biopsies was 93.5% (146/156). All three major complications occurred in chest biopsies. The median total radiation dose was 522.4 (393.4–819.8) mGy*cm. Of the total radiation dose, 87% was applied during the pre- and post-interventional CT series. Post-interventional CT accounted for 24.4% of the total radiation dose. Only 11.4% of the dose was applied by CTF-guided intervention. Multilinear regression demonstrated that male sex, body mass index, drainage, intervention time, and helical scan as post-interventional CT were significantly associated with higher dose.Conclusion:The 320-detector row CTF interventions achieved a high success rate. Dose reduction in post-interventional CT provides patient dose reduction without decreasing the technical success rates.Advances in knowledge:This is the first study on the relationship between various interventional outcomes and patient exposure to radiation in 320-detector row CTF-guided interventions, suggesting a new perspective on dose reduction.     

Assessment of Hounsfield unit in the differential diagnosis of odontogenic cysts

Objectives:The purpose of this study was to evaluate the usefulness of Hounsfield unit (HU) assessment with multislice-CT in the differentiation of radicular cysts (RCs), dentigerous cysts (DCs) and odontogenic keratocysts (OKCs).Methods:In total, 307 odontogenic cysts (RCs, DCs and OKCs) were included in this study. Cysts with lesion diameter <10 mm, cysts with artefacts affecting measurement of HU values, cysts involving infection and recurrent cysts were regarded as exclusion criteria. Images were acquired in three different types of CT scanners: Aquilion ONE, Discovery CT750 HD and SOMATOM Definition Flash. Differences in HU values among scanners and among types of odontogenic cysts were assessed using one-way analysis of variance; multiple comparisons were performed post hoc, using the Tukey–Kramer honestly significant difference test.Results:In total, 164 cysts were analysed in this study (64 RCs, 57 DCs and 43 OKCs). Regardless of the type of lesion, the Aquilion ONE scanner demonstrated a significant difference in HU value, compared with the Discovery CT750 HD scanner. Regardless of CT scanner model, HU values significantly differed between DCs and OKCs (p < 0.0001), as well as between OKCs and RCs (p < 0.0001).Conclusions:HU values were found to vary among CT scanners and should always be associated with other lesion imaging features while interpreting and elaboration diagnostic hypothesis. Notably, the results suggested that OKCs might be able to be differentiated from DCs and RCs by using HU values.     

Evaluation of dimensional accuracy and degree of polymerization of stereolithography photopolymer resin under different postpolymerization conditions: An in vitro study

Statement of problemThe appropriate postpolymerization of stereolithography (SLA) resins with the least effect on dimensional accuracy and with optimal polymerization is unclear.PurposeThe purpose of this in vitro study was to investigate the dimensional accuracy and degree of polymerization of a photopolymer resin for SLA with different postpolymerizing times and temperatures.Material and methodsSixty 1.5-mm-thick specimens were made from clear photopolymer resin with a 3D printer to simulate a maxillary complete denture. They were postpolymerized for different periods (15 and 30 minutes) at 3 different temperatures (40 °C, 60 °C, and 80 °C). Both prepolymerization and postpolymerization gap sizes for each specimen were measured at 5 different locations under a stereomicroscope. The tissue surface was scanned before and after polymerization, and the images were superimposed. The deviation was analyzed by using computer-aided design (CAD) software; root mean square estimates (RMSE) and color map data were obtained. Fourier transform infrared spectrometry was used to determine the degree of conversion (DC) of all specimens. The Kruskal-Wallis and Mann-Whitney tests were used to calculate the difference value of the gap sizes (α=.05). One-way ANOVA and the Tukey test were used for RMSE and DC (α=.05).ResultsThe smallest average change in gap sizes was found at 15 minutes and 40 °C, and the largest change at 30 minutes and 80 °C. The lowest RMSE was obtained at 30 minutes and 40 °C (P<.05). On the color map, a uniform deposited layer was created at 15 minutes and 40 °C and 30 minutes and 40 °C. The highest DC was found at 30 minutes and 60 °C, which differed significantly from 15 minutes and 40 °C (P<.05). The lowest degree of polymerization was found at 30 minutes and 40 °C.ConclusionsThe polymerizing temperature exerted a greater effect than polymerizing time, with lower temperatures leading to improved fit and tissue surface accuracy. The recommended parameters for SLA polymerization are 15 minutes and 40 °C. These conditions offered high dimensional accuracy, favorable surface tissue adaptation, and satisfactory DC.     

Initial report of bridge to recovery in a patient with DuraHeart LVAD

Continuous-flow left ventricular assist devices (LVADs) provide acceptable clinical results, but the long waiting period for heart transplantation leads to diverse complications. LVAD support can cause reverse left ventricular (LV) remodeling that results in the improvement of LV function and allows LVAD removal. We present a case of successful removal of a DuraHeart LVAD because of sufficient recovery of LV function. Before LVAD removal, we conducted an “LVAD weaning test” by decreasing pump speed and performing an additional normal saline infusion test. We consider that the LVAD weaning test can be used in place of the “pulsatile LVAD off test.”     

Suture Granuloma With False-Positive Findings on FDG-PET/CT Resected via Laparoscopic Surgery

A 61-year-old woman who had undergone total hysterectomy 16 years previously exhibited a pelvic tumor on computed tomography (CT). F-18 fluorodeoxyglucose (FDG) combined positron emission tomography (PET)/CT imaging revealed a solitary small focus of increased FDG activity in the pelvis. A gastrointestinal stromal tumor originating in the small intestine or another type of tumor originating in the mesentery (desmoid, schwannoma, or foreign body granuloma) was suspected; therefore, laparoscopic resection was conducted. A white, hard tumor was found to originate from the mesentery of the sigmoid colon and adhered slightly to the small intestine. The tumor was resected with a negative margin, and the pathologic diagnosis was suture granuloma. The possibility of suture granuloma should be kept in mind in cases of tumors with positive PET findings and a history of surgery close to the lesion. However, it is difficult to preoperatively diagnose pelvic tumors using a biopsy. Therefore, considering the possibility of malignancy, it is necessary to achieve complete resection without exposing the tumor.Key words: Suture granuloma, Laparoscopy, Positron emission tomography (PET)It is very difficult to diagnose suture granulomas preoperatively. F-18 fluorodeoxyglucose (FDG) combined positron emission tomography (PET)/computed tomography (CT) imaging is often used to differentiate benign from malignant tumors that are difficult to diagnose on other modalities, such as ultrasound (US), CT, and magnetic resonance imaging. However, it is not easy to differentiate tumors associated with inflammation or malignancy using FDG-PET/CT. Suture granulomas are known to be benign; however, false-positive findings were observed on PET/CT in our case. In the literature, there are few reports of suture granulomas showing false-positive findings on PET/CT.^1–5 We report here a case in which it was not possible to rule out the potential for malignancy using CT or FDG-PET/CT and the lesion was confirmed to be a suture granuloma based on a pathologic examination following laparoscopic resection.