Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts

Aim:

Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I_to) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods:

Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results:

Neferine (0.1–100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC₅₀ value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD₁₀ at Epi sites.

Conclusion:

Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD₁₀ at Epi sites of rabbit heart.     

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

Aim:

To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target.

Methods:

α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.

Results:

Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel.

Conclusion:

We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.     

Mesoridazine: an open-channel blocker of human ether-a-go-go-related gene K+ channel

Mesoridazine, a phenothiazine antipsychotic agent, prolongs the QT interval of the cardiac electrocardiogram and is associated with Torsade de pointes-type arrhythmias. In this study, we examined the effects of mesoridazine on human ether-a-go-go-related gene (HERG) K+ currents. HERG channels were stably expressed in human embryonic kidney 293 cells and studied using standard whole-cell patch-clamp technique (37 degrees C). Mesoridazine blocked HERG currents in a concentration-dependent manner (IC50 550 nM at 0 mV); block increased significantly over the voltage range where HERG activates and saturated at voltages eliciting maximal HERG channel activation. Tonic block of HERG current by mesoridazine (1.8 microM) was minimal (< 2-4%). The rate of the onset of HERG channel block was rapid and dose dependent (tau = 54 +/- 7 ms at 0 mV and 1.8 microM mesoridazine), but not significantly affected by test potentials ranging from -30 to +30 mV. The V1/2 for steady-state activation was shifted from -31.2 +/- 1.0 to -39.2 +/- 0.5 mV (P < 0.01). The apparent rate of HERG channel deactivation was significantly reduced (fast tau = 153 +/- 8 vs. 102 +/- 6 ms at -50 mV, P < 0.01; slow tau = 1113 +/- 63 vs. 508 +/- 27 ms, P < 0.01). The inactivation kinetics and voltage dependence of steady-state inactivation of the HERG channel were not significantly altered by mesoridazine. These findings demonstrate that mesoridazine is a potent and rapid open-channel blocker of HERG channels. This block would explain the QT prolongation seen clinically at therapeutic concentrations (0.3-3.6 microM).     

Dissociation between sleep-related and TRH-induced prolactin secretion in seminiferous tubule failure

Prolactin (PRL) secretion has been measured during sleep and following TRH administration in 8 patients aged 24-39 yr with seminiferous tubule failure and 36 controls. Basal LH levels were 25.7 +/- 14.7 mIU/ml in the patients compared to 11.5 +/- 4.2 mIU/ml in the controls (p less than 0.01) Corresponding FSH levels were 26.2 +/- 10.7 mIU/ml and 5.9 +/- 2.1 mIU/ml (p less than 0.001) Mean estradiol 17B and testosterone levels were similar in the 2 groups. The mean PRL secretion during sleep was 16.5 +/- 11.7 ng/ml in the patients and not different in 11 of the controls (12.4 +/- 3.2 ng/ml). One patient had a mean nocturnal PRL concentration of 44.1 ng/ml. In both groups, the mean sleep related PRL concentration was greater than that during waking hours. The average number of peaks in the 2 groups was similar. In the same patients, the peak PRL response to TRH (200 ug IV) was 81.9 +/- 18.8 ng/ml as compared to 32.1 +/- 10.7 ng/ml in the controls (p less than 0.001). It is concluded that PRL concentrations following pharmacological stimulation are increased in seminiferous tubule failure, whereas levels are normal in relation to the physiological stimulus of sleep.     

卡维地洛对RyR2介导的自发性钙振荡的抑制作用

 目的：探讨并比较卡维地洛和其它β受体阻滞剂对兰尼定受体2（RyR2）介导的自发性钙振荡的抑制作用。方法：采用Flp-In T-REx Core Kit构建可以稳定诱导表达RyR2的HEK293细胞株,采用酶解方法分离大鼠单个心肌细胞。逐步提高细胞外钙离子浓度,诱发细胞自发性钙振荡。单细胞钙影像技术记录并检测卡维地洛等β受体阻滞剂对HEK293细胞株及心肌细胞自发性钙振荡的作用。结果：卡维地洛30 μmol/L可明显抑制心肌细胞和表达RyR2的HEK293细胞株的自发性钙振荡,其抑制率分别为(6530±230)%和(6908±530)%;而美托洛尔、索他洛尔、阿替洛尔等常用的14种β受体阻滞剂对心肌细胞及HEK293细胞株的自发性钙振荡均无明显作用。结论：卡维地洛是唯一可以抑制RyR2介导的自发性钙释放的β受体阻滞剂,这可能是卡维地洛在降低心衰死亡率上优于其它β受体阻滞剂的机制之一。     

蛋白磷酸酶2A催化亚基下调参与人tau蛋白所致线粒体分裂融合和功能失衡

目的 探讨蛋白磷酸酶 2A 催化亚基(PP2Ac)下调是否参与人tau蛋白积聚引起的线粒体分裂融合动态及功能失衡的机制。方法 大鼠原代海马神经元转染mito-dsRed质粒和人tau40质粒48 h后,共聚焦显微镜观测线粒体分布,免疫印迹检测线粒体分裂融合蛋白、PP2Ac 及PP2A 调节亚基 b(PP2Ab)表达水平;大鼠原代海马神经元和野生型 HEK293 细胞(293wt)共转染siPP2Ac-EGFP质粒和mito-dsRed质粒48 h后,检测线粒体形态和分布;293wt细胞共转染siPP2Ac质粒和mito-Dendra2质粒40 h后,实时观测线粒体分裂融合动态;293wt 细胞沉默 PP2Ac表达后,检测线粒体分裂融合蛋白水平及细胞膜电位和细胞活力;稳定表达人tau的HEK293细胞(293htau)共转染PP2Ac-EGFP质粒和mito-dsRed质粒48 h后,分析线粒体分布和形态及功能。结果 大鼠原代海马神经元表达人 tau 蛋白引起突起线粒体分布下降并伴有PP2Ac水平显著下降(t=4.814, P=0.0086)。下调PP2Ac表达引起大鼠原代海马神经元和HEK293细胞线粒体变长并异常分布于细胞核周围;引起293wt细胞线粒体融合明显加速(t=2.857, P=0.0074);使293wt细胞融合蛋白线粒体融合蛋白(MFN)1(t=6.768, P=0.0025)、MFN2(t=3.121, P=0.0035)和视神经萎缩蛋白1水平显著升高(t=3.775, P=0.0199),动力样蛋白1和Fis1水平不变;使HEK293细胞线粒体相对膜电位(t=2.300, P=0.0270)和细胞活力(t=6.249, P<0.0001)显著降低。上调PP2Ac表达可减轻293htau细胞线粒体形态和分布及功能异常。结论 PP2Ac表达下调参与了人tau蛋白引起的大鼠原代海马神经元和HEK293细胞线粒体形态分布异常及细胞活力下降。     

毕赤酵母表达重组人凝血酶原2及活性分析

目的：通过毕赤酵母（Pichia pastoris）表达制备重组人凝血酶原2（prothrombin-2）,并利用锯鳞蝰（Echis carinatus）凝血酶原激活剂ecarin将其活化为凝血酶,分析凝血酶的活性。方法根据GenBank公布的人凝血酶原2和ecarin的cDNA序列,设计并优化合成凝血酶原2和ecarin基因。将凝血酶原2基因构建至表达载体pPICZαA中,转化并筛选重组凝血酶原2的糖基工程酵母菌,诱导工程酵母分泌表达凝血酶原2,培养上清中的目的蛋白经两步阳离子层析纯化制备,并利用酶切N-糖链和肽指纹图谱分析鉴定目的蛋白。将ecarin基因构建到表达载体pcDNA3．1（＋）,瞬转HEK 293T细胞,收集培养上清。将HEK 293T工程细胞培养上清与纯化的凝血酶原2反应,利用凝血酶发色底物S-2238,测反应产物的酶促活性;利用血浆纤维蛋白原测反应产物的血凝时间、分析血凝活性。结果酵母表达凝血酶原2的培养上清经纯化获得37×103的目的蛋白,去除N-糖链的蛋白相对分子质量降为35×103,与凝血酶原2理论分子量一致。经肽指纹图谱鉴定,纯化得到的蛋白为凝血酶原2。制备的凝血酶原2经HEK 293T细胞表达ecarin的培养上清处理后,可催化S-2238解离产生黄色的对硝基苯胺（pNA）,且pNA产生的光密度值随着处理的凝血酶原2的增加而升高;经ecarin处理的凝血酶原2能促进血浆凝结,与凝血酶试剂的血凝时间接近,且血凝时间随着处理的凝血酶原2的量减少而延长。结论利用毕赤酵母制备了重组人凝血酶原2,被ecarin活化为具有活性的凝血酶,有望替代从血浆中提取的凝血酶原,用于战伤救治和临床研究。     

利用Crispr/Cas9构建POMP基因组原位荧光报告系统及其应用

目的 利用Crispr/Cas9系统建立蛋白酶体成熟蛋白(proteasome maturation protein,POMP)基因组原位荧光报告系统.方法 构建POMP-2A-mCherry-T载体并转染HEK293(human embryonic kdiney 293 cell)细胞;流式细胞仪分选获取mCherry阴阳性细胞;实时荧光定量PCR(real-time fluorescent quantitative PCR)检测mCherry阴阳性细胞中POMP基因转录水平表达差异;Western blot检测mCherry阴阳性细胞中POMP蛋白表达差异以及Huh7细胞中JNK1/JNK2磷酸化水平;蛋白酶体活性试剂盒检测mCherry阴阳性细胞中蛋白酶体活性差异.结果 mCherry阳性细胞POMP蛋白表达水平比mCherry阴性细胞高1.3倍(P＜0.01),但其基因转录水平差异并不显著(P＞0.05);同时mCherry阴性细胞中泛素化水平比mCherry阳性细胞高1.2倍(P＜0.01);另外mCherry阳性细胞中蛋白酶体活性也显著高于阴性细胞(P＜0.01);低表达POMP的Huh7细胞中,JNK1和JNK2的磷酸化水平分别升高1.5倍和1.4倍(P＜0.01).结论 成功构建POMP荧光报告系统;将该系统用于Huh7细胞,发现POMP可改变JNK1、JNK2磷酸化水平,其可能通过JNK信号通路来影响细胞的增殖以及凋亡.     

Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W

Congenital stationary night blindess-2 (incomplete congenital stationary night blindness (iCSNB) or CSNB-2) is a nonprogressive, X-linked retinal disease which can lead to clinical symptoms such as myopia, hyperopia, nystagmus, strabismus, decreased visual acuity, and impaired scotopic vision. These clinical manifestations are linked to mutations found in the CACNA1F gene which encodes for the Ca(v)1.4 voltage-gated calcium channel. To better understand the physiological effects of these mutations, three missense mutants, F742C, G1007R and R1049W, previously shown to be mutated in patients with CSNB-2, were transiently expressed in human embryonic kidney (HEK) tsA-201 cells and characterized using whole-cell patch clamp. The G1007R mutation is located in transmembrane segment 5 (S5) of domain III and R1049W is located in the extracellular linker between S5 and the P-loop of domain III. Both mutants produced full length proteins that targeted to the membrane but did not support ionic currents. In 20 mM Ba(2+), F742C (S6 domain II) produced a approximately 21 mV hyperpolarizing shift in half activation potential (V(a[1/2])) and a approximately 23 mV hyperpolarizing shift in half inactivation potential (V(h[1/2])). Additionally, F742C displayed slower inactivation kinetics and a smaller whole cell conductance (G(max)). In physiological 2 mM Ca(2+), F742C produced a approximately 19 mV hyperpolarizing shift in V(a[1/2]). These findings suggest that the pathology of CSNB-2 in patients with these missense mutations in the Ca(v)1.4 calcium channel is the result in either a gain of function (F742C) or a loss of function (G1007R, R1049W).     

基因治疗原发性高血压的新途径:内皮型一氧化氮合成酶CDNA腺病毒载体在人胎肾293细胞的表达

研究内皮型一氧化氮合成酶（endothelialnitric oxide synthase,eNOS)CDNA 腺病毒载体 PadRSVeNOSCDNA 导入在人胎肾 293细胞 ( )中的表达。方法:大量制备及纯化质粒 eNOSCDNA ,将纯化eNOSCDNA 载体通过脂质体转染 293细胞48h 后,应用化学比色法测定293细胞提取物NOS 活性。结果:被转染 293细胞 NOS活性显著提高于未转染293细胞 NOS 活性犤( 0.62±0.03),(0.093±0.01)kU /L,P <0.001犦。结论:PA dRSVeNOSCDNA 导入人胎肾 293细胞是一种能使 NOS 活性显著升高的方法,为基因治疗原发性高血压及肾脏血管性疾病研究提供一条新的途径。