Effects of redox potential control on succinic acid production by engineered Escherichia coli under anaerobic conditions

The effects of oxido-reduction potential (ORP) control on succinic acid production have been investigated in Escherichia coli LL016. In LL016, two CO₂ fixation pathways were achieved and NAD⁺ supply was enhanced by co-expression of heterologous pyruvate carboxylase (PYC) and nicotinic acid phosphoribosyltransferase (NAPRTase). During anaerobic fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of −200 to −400 mV. From the results, the ORP level of −400 mV was preferable, which resulted in the high succinic acid concentration (28.6 g/L) and high succinic acid productivity (0.33 g/L/h). Meanwhile, the yield of succinic acid at the ORP level of −400 mV was 39% higher than that at the ORP level of −200 mV. In addition, a higher NADH/NAD⁺ ratio and increased enzyme activities were also achieved by regulating the culture to a more reductive environment, which further enhanced the succinic acid production.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

Influence of redox potential on methanation of methanol by Methanosarcina barkeri in Eh-stat batch cultures

The effect of Eh on the methanogenesis of methanol by Methanosarcina barkeri strain Fusaro was studied in pH-controlled anaerobic batch cultures at 37°C, in which the Eh of the culture medium was controlled by the addition of Ti(III)-citrate at values ranging from −340 to −520 mV. The changes in Eh revealed that the specific growth rate, μ, specific methane production rate, Q_CH4 and growth yield, Y_X/S were optimum under an Eh between −430 and −520 mV, while they decreased at the higher Eh of −340 mV. The maximum values of Q_CH4 and μ under the optimum Eh condition were 210 ml CH₄/g dry cell weight·h⁻¹ and 0.11 h⁻¹, respectively.     

Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody,an Equine Standard and a Human Tracer

A radioimmunoassay for llama and alpaca LH was developed using a human I125LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L−1. The intra-assay coefficient of variation was 37% at 1 μg L−1, declined to 15% at 4 pg L−1 and was below 6% for concentrations up to 32 μg L−1. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L−1), 16% (7.1 μg L−1) and 9.8% (19 μg L−1). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L−1. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).     

Tolbutamide-sensitive potassium conductance in the basolateral membrane of A6 cells

K⁺ channels sensitive to intracellular ATP (K_ATP channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K⁺ channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 ± 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of ?83 ± 1 mV and was inward rectifying in spite of the outwardly directed K⁺ gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 ± 3.0 μm and 0.114 ± 0.018 μm for tolbutamide and glibenclamide respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 mm) and insulin (250 μU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K⁺ conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP.     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

硫胺素对Arthrobacter sp.A302环磷酸腺苷发酵的影响

研究在培养基中加入硫胺素（V B1）对cAMP发酵的影响。结果表明：V B1的最适添加量为0.5 g/L,与对照组相比,环磷酸腺苷（cAMP）产量和细胞干质量分别提高了36.4%和41.8%,达到7.5和7.8 g/L。琥珀酸和α-酮戊二酸的含量有明显的提高,平均提高了43.59%和40.77%;同时,主要副产物乙酸的含量没有明显变化。在发酵过程中,V B1的加入对ATP的含量及与cAMP合成密切相关的几种酶的活性也有显著的影响。     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Adaptive evolution improves acid tolerance and succinic acid production in Actinobacillus succinogenes

Fermentation at low pH is an efficient way to improve the competitiveness of biological succinic acid-producing process. Actinobacillus succinogenes shows good performance of succinic acid production under anaerobic conditions, but its succinic acid production capability at the low-pH is inefficient due to the poor acid resistance. Herein, a mutant A. succinogenes BC-4 with improved cell growth and succinic acid production under weak acid conditions was obtained by adaptive evolution. The specific growth rate and succinic acid production of BC-4 reached 0.13 g/L/h and 20.77 g/L, which were increased by 3.25- and 2.95- fold, respectively compared with the parent strain under anaerobic condition at pH 5.8. The activities of specific enzymes with ATP generation were significantly enhanced under weak acidic conditions, resulting in 1.28-fold increase in the maximum ATP level. Membrane fatty acid composition analysis demonstrated that the ratio of saturated to unsaturated fatty acids was decreased from 1.62 to 1.44 in mutant BC-4, leading to improved intracellular pH homeostasis. Furthermore, the change from long-chain to median-chain fatty acid might lower the permeability of H⁺ into cytoplasm for survival under acid stress. These results indicated that A. succinogenes BC-4 is a promising candidate for succinic acid production under weak acid condition.     

Characterization of temperature-sensitive mutants of animal cells

An early increase in lymphocyte plasma membrane K⁺ transport is essential for PHA stimulated lymphocytes to divide. Little is known about the specific source and amount of energy required to support the increased transport by activated lymphocytes. Since ouabain, a cardiac glycoside, specifically inhibits the transport ATPase, we have measured the decrement in glycolysis and tricarboxylic acid cycle activity when untreated and PHA treated lymphocytes were exposed to ouabain. This metabolic decrement represents the portion of metabolism associated with monovalent cation transport and closely related processes. Since TCA cycle activity accounted for only 0.2% of glucose consumption, aerobic glycolysis was the major source of energy, i.e., ATP, for increased transport. Approximately one-third of the total lactate production in both control and PHA stimulated lymphocytes was ouabain-sensitive. Ouabain sensitive lactate production in control, 105 μmol/10¹⁰ cells/hour, increased 1.8-fold to 193 μmol/10¹⁰ cells/hour after PHA treatment. Active K⁺ influx in similar cell populations increased from 40 μmol/10¹⁰ cells/hour to 74 μmol/10¹⁰ cells/hour (1.9-fold) after PHA treatment. The increment in ouabain-sensitive energy production and K⁺ transport were closely correlated and, therefore, 0.38 moles of K⁺ are transported for each mole of ATP generated in both control and PHA treated cells. The increased requirement for transport related energy is provided by increasing the ouabain-sensitive ATP production rather than altering the efficiency of ATP transduction.     

Methanogenesis from trimethylamine + H2 by Methanosarcina barkeri is coupled to ATP formation by a chemiosmotic mechanism

Cell suspensions of Methanosarcina barkeri catalyzed the conversion of trimethylamine and molecular hydrogen to methane according to the equation (CH₃)₃NH⁺ + 3 H₂ → 3 CH₄ + NH⁺₄. The onset of methane formation resulted in an increase of the intracellular ATP content from 2 to 4.6 nmol/mg protein and in the generation of a protonmotive force (Δp) of −130 mV, of which the Δψ contributed 90%. The addition of the uncoupler led to a drastic decrease of the intracellular ATP content and the Δψ, but stimulated methanogenesis. The ATPase inhibitor DCCD caused a rapid exhaustion of the ATP pool and inhibited methane formation, whereas Δψ was not affected. The inhibition of methane formation by DCCD could be relieved by addition of TCS, indicating a chemiosmotic coupling between methane formation according to the above equation and ATP synthesis.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

精胺对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

研究了在添加外源精胺时,霍山石斛类原球茎细胞生长、多糖积累、主要营养物质消耗以及细胞内多胺含量的变化。结果表明,0.6mmol/L的精胺明显促进霍山石斛类原球茎细胞的生长和多糖的合成。细胞的比生长速率从0.046d-1提高到0.054d-1。培养30d时,类原球茎干重达32.4gDW/L,多糖总产量为2.46g/L,分别是对照的1.32和1.31倍。添加外源精胺能够提高内源多胺的含量,同时,蔗糖酶和硝酸还原酶等相关代谢酶的活性增强,促进了碳、氮的吸收和利用。     

精胺对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

研究了在添加外源精胺时,霍山石斛类原球茎细胞生长、多糖积累、主要营养物质消耗以及细胞内多胺含量的变化。结果表明,0.6mmol/L的精胺明显促进霍山石斛类原球茎细胞的生长和多糖的合成。细胞的比生长速率从0.046d^-1提高到0.054d^-1。培养30d时,类原球茎干重达32.4g DW/L,多糖总产量为2.46g/L ,分别是对照的1.32和1.31倍。添加外源精胺能够提高内源多胺的含量,同时,蔗糖酶和硝酸还原酶等相关代谢酶的活性增强,促进了碳、氮的吸收和利用。     

Ethanol perfusion increases the yield of oxidative phosphorylation in isolated liver of fed rats

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria.The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using ³¹P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O₂ Clark electrodes.The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86±0.02 μmol/min g wet weight (min g ww)] than in control [1.44±0.18 μmol/min g ww]. However, mitochondrial respiration remained unchanged [2.20±0.13 μmol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33±0.035 (control) to 0.42±0.015 (ethanol).The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.     

Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

Increase of xylitol production rate by controlling redox potential in Candida parapsilosis

The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L. h to 4.62 g/L. h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L. h.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Association of lead and cadmium exposure with kidney stone incidence: A study on the non-occupational population in Nandan of China

BackgroundEnvironmental lead (Pb) and cadmium (Cd) pollution has been considered a risk factor in the etiology of kidney stones. However, the association between Pb and Cd exposure and kidney stone incidence has yet to be determined.ObjectivesThis study aimed to determine a possible the association between kidney stones with Pb and Cd exposure (alone or combined) in a non-occupational population.MethodsPb and Cd contaminations in soil-plant system were determined by flame atomic absorption spectrophotometry. Health risk assessment of dietary Pb or Cd intake from rice and vegetables were calculated. Kidney stones were diagnosed with urinary tract ultrasonography. Urinary cadmium (UCd) and blood lead (BPb) levels were determined by graphite-furnace atomic absorption spectrometry. Multivariate logistic regression models were constructed.ResultsThe hazard indexes (HI) of Pb and Cd were 7.91 and 7.31. The odds ratio (OR) was 2.83 (95 %CI:1.38−5.77) in males with high BPb (BPb ≥ 100 μg/L), compared with those with low BPb (BPb＜100 μg/L). Compared to those with low BPb and low UCd (BPb＜100 μg/L and UCd＜2 μg/g creatinine), the ORs were 2.58 (95 % CI:1.17−5.70) and 3.43 (95 % CI:1.21−9.16) in females and males with high BPb and high UCd (BPb ≥100 μg/L and UCd ≥2 μg/g creatinine), respectively. The OR was 3.16 (95 % CI:1.26−7.88) in males with high BPb and low UCd (BPb ≥ 100 μg/L and UCd ＜2 μg/g creatinine), compared to those with low BPb and low UCd.ConclusionsKidney stones incidence was increased by high Pb exposure in males, and by Pb and Cd co-exposure in males and females.     

Mg(2+) inhibits ATP-activated current mediated by rat P2X4 receptors expressed in Xenopus oocytes

To investigate the modulation of Mg(2+) on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg(2+) on ATP-activated currents (I(ATP)) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg(2+) on I(ATP) were as follows: (1) In oocytes expressing P2X4 receptors, Mg(2+) with concentration ranging from 0.5-10 mmol/L inhibited the amplitude of I(ATP) in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC(50)) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg(2+) (1 mmol/L) shifted the dose-response curve for I(ATP) right-downward without changing the EC(50), but reduced the maximal current (E(max)) by (42.0 ± 2.1)%. (3) After being preincubated with Mg(2+) for 80 s, the inhibitory effect of the Mg(2+) on I(ATP) reached the maximum. (4) The inhibition of Mg(2+) on I(ATP) was independent of membrane potential from -120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of I(ATP) was increased almost one-fold, and Mg(2+) with concentration ranging from 0.5-10 mmol/L did not affect the I(ATP) significantly. The results suggest that Mg(2+) inhibits I(ATP) mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg(2+) might be realized by acting on the site Asp280 of the P2X4 receptors.