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91.
92.
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, 79) and are involved in many physiological and pathophysiological processes (1012). 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 (2026), 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 (3234) 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   
93.
目的 建立一种丸剂中非法添加环丙沙星的快速检测方法。方法 通过19F-核磁共振定量(19F-qNMR)法对添加入当归苦参丸中的环丙沙星进行检测,以4,4’-二氟二苯甲酮为内标,以氘代DMSO为溶剂,采集δ-120.3处环丙沙星信号和δ-104.8处内标信号面积,计算环丙沙星的含量。结果 以环丙沙星和内标信号面积比为纵坐标,环丙沙星和内标质量比为横坐标,得到线性回归方程为:Y=3.384 1X+0.114 8,R2=0.992,环丙沙星在5~30 mmol/L具有良好的线性,检出限为7.798×10-2 mg/mL,定量限为0.260 mg/mL。建立的19F-qNMR法精密度、重复性、稳定性、加样回收率均符合检测要求。结论 所建立的19F-qNMR法样品制备简单,检测时间短,灵敏度高,适合对丸剂中非法添加的环丙沙星进行含量测定。  相似文献   
94.
芹菜素γ-环糊精包合物的表征和抗氧化活性研究   总被引:1,自引:0,他引:1  
目的研究芹菜素与γ-环糊精(γ-CD)包合物的制备、表征、包合机制及其抗氧化活性。方法采用加热回流共沉淀法制备芹菜素γ-环糊精包合物,红外光谱法(IR)对形成的包合物进行表征,荧光光谱法测定包合物的包合常数(K)和包合比(n);荧光光谱法和核磁共振法(NMR)研究芹菜素与γ-CD在溶液中形成的包合物;双倒数曲线法研究包合前后对自由基1,1-二苯基-2-三硝基苯肼(DPPH·)的清除率。结果在不同的pH值溶液中,γ-CD对芹菜素具有不同的包合能力,γ-CD最适合在中性介质中与芹菜素形成包合物,在实验浓度范围内按1∶1形成包合物,包合比为1236,包合物对自由基DPPH·的清除活性更强;NMR确定包合物的结构。结论在实验条件下,芹菜素分子从γ-CD大口端进入到空腔内形成稳定的包合物,且抗氧化活性增加。  相似文献   
95.
96.
In HIV type 1 (HIV-1), the dimerization initiation site (DIS) is the sequence primarily responsible for initiating the noncovalent linkage of two homologous strands of genomic RNA during viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence and forms a symmetric homodimer through a loop-loop kissing interaction. In a structural rearrangement catalyzed by the HIV-1 nucleocapsid protein (NCp7) and suggested to be associated with maturation of the budded viral particle, the DIS converts from a metastable kissing dimer to an extended duplex. Here, we demonstrate that the DIS kissing dimer displays localized conformational dynamics that result from the specific protonation of the N1 base nitrogen of the DIS loop residue A272 at near-physiological pH. The rate of NCp7-catalyzed maturation of the DIS kissing dimer is also shown to directly correlate with the observed proton-coupled conformational dynamics, where NCp7 is found to convert the dynamic A272 protonated state with a faster rate. Taken together, these results reveal a previously undescribed role for base protonation in modulating local RNA structure and demonstrate a mechanism for promoting the chaperone-mediated structural rearrangement of a kinetically trapped RNA conformational state.  相似文献   
97.
Potassium channel opener's (KCOs) were originally thought of as nonselective smooth muscle relaxants. However, recent investigations in animal models of both peripheral vascular disease (PVD) and asthma have revealed interesting effects of these drugs at unexpectedly low doses. Hemodynamically, KCOs are interesting in PVD since they have little effect on blood supply to normally perfused skeletal muscle, but enhance perfusion to chronically ligated ischemic tissue. In animal PVD models, SDZ PCO-400 and cromakalim have been shown to improve recovery of muscle energy stores from ischemia or to preserve performance under conditions of ischemic contracture. Beneficial effects in rat PVD models were manifest at doses below those affecting systemic blood pressure and may be attributable to a selective dilatation of collateral vessels. With regard to the airways, the apparent efficacy of KCOs as antiasthmatic drugs seems not to be attributable solely to their bronchodilator activity. Although KCOs elicit no antiinflammatory effect in animal models, studies with SDZ PCO-400 in guinea pigs sensitized to antigen or treated with immune complexes have revealed that expression of airway hyperreactivity is significantly inhibited at drug doses exhibiting only modest bronchodilator activity. At least part of this action can be attributed to inhibition at the level of neural innervation of the airways, possibly through attenuation of nonadrenergic noncholinergic (NANC) transmission. Thus, based on results generated in animal models of asthma and PVD, clinical evaluation of the KCOs in these indications would seem warranted, with the hope that (due to their selective actions) beneficial therapeutic effects can be achieved at doses devoid of unwanted systemic actions.  相似文献   
98.
One hundred and twenty excised rabbits hearts were subjected to 1 h of continuous pulsatile coronary perfusion with acellular fluids in a Lindbergh-Rockefeller Institution organ perfusion apparatus. Perfusions were carried out at 26°C and 15°C. At the end of 1 h of perfusion, samples of the left ventricular myocardium were packed into nuclear magnetic resonance (NMR) tubes and the water peak area and linewidth of the water signal were determined by the steady-state NMR spectroscopy. Seventeen different perfusates were employed. Four hearts were perfused and one control heart was processed in an identical way, with each set of experiments. Results were expressed as the mean percentage increase in total myocardial water in perfused hearts, as compared to unperfused hearts. At 26°C these ranged from 9.31 (s.e. 0.66) to 35.89 (s.e. 4.09). At 15°C the range was from 13.26 (s.e. 1.03) to 39.14 (s.e. 2.06). At 26°C, the mean linewidth (Hz) in control myocardiums was 11.60 (s.e. 0.40), in perfused hearts 7.34 (s.e. 0.33); at 15°C it was 12.15 (s.e. 0.29) in control hearts, and 7.84 (s.e. 0.37) in perfused ones. Hearts perfused with lactated Ringer's solution showed the greatest interstitial water accumulation. Linewidth narrowing indicated an accumulation of “unstructured” presumably extracellular water. In edematous hearts, interstitial spaces were widely dilated. The described technique can serve as a rapid screening method for assessing myocardial edema produced by perfusion.  相似文献   
99.
目的:通过离体的高分辨核磁共振氢谱(1H NMR)技术研究2型糖尿病db/db小鼠小脑的代谢变化。方法:采用8只15周龄2型糖尿病模型db/db小鼠为实验组,11只15周龄野生正常小鼠为对照组,对其小脑组织进行离体的1H NMR检测分析。结果:实验组与对照组相比小脑的代谢模式明显不同,甘氨酸(Gly)、天冬氨酸(Asp)、谷氨酸(Glu)、γ-氨基丁酸(GABA)、N-乙酰天门冬氨酸(NAA)、丙氨酸(Ala)等代谢物浓度明显下降,差异有统计学意义(P<0.05或P<0.01);谷氨酰胺(Gln)、牛磺酸(Tau)、乳酸(Lac)等代谢物水平显著上升,差异有统计学意义(P<0.05或P<0.01)。结论:代谢组学结果说明2型糖尿病模型db/db小鼠小脑中糖代谢、氨基酸代谢出现了紊乱,这为进一步认识糖尿病脑病的发病机制提供了线索。  相似文献   
100.
BACKGROUND & AIMS: Biofeedback is reported to be as effective for slow transit constipation as for pelvic floor dyssynergia and no more effective than education. We aimed to test the hypothesis that biofeedback benefits only patients with pelvic floor dyssynergia, describe the physiologic mechanism of treatment, and identify predictors of success. METHODS: Fifty-two patients (49 women; average age, 35 years), all with delayed whole gut transit, included 34 with pelvic floor dyssynergia, 12 with slow transit only, and 6 who met only 1 of 2 criteria for pelvic floor dyssynergia. All received 5 weekly biofeedback sessions directed at increasing rectal pressure and relaxing pelvic floor muscles during straining plus practice defecating a balloon. Patients were retested by questionnaire; symptom diary; balloon defecation; transit study at 1, 6, 12, and 24 months; and anorectal manometry at 1 and 6 months. RESULTS: At 6 months, greater improvements were seen in pelvic floor dyssynergia compared with slow transit only; 71% versus 8% reported satisfaction ( P = .001), and 76% versus 8% reported >/=3 bowel movements per week ( P < .001). Improvements were maintained at 24 months of follow-up. Biofeedback eliminated dyssynergia in 91% and enabled 85% to defecate the balloon. Satisfaction was correlated with improved ability to defecate the balloon (rho = .73; P < .001), reductions in dyssynergia (rho = .69; P < .001), and increased rectal pressure during straining (rho = .36; P < .01). Success was predicted by pelvic floor dyssynergia, milder constipation, and less frequent abdominal pain at baseline. CONCLUSIONS: Biofeedback is an effective treatment for pelvic floor dyssynergia but not slow transit constipation.  相似文献   
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