Notch信号通路在肺癌干细胞中的表达及其对增殖的影响

目的 观察Notch信号通路在肺癌干细胞中的表达及阻断Notch信号通路对肺癌干细胞增殖的影响.方法 以CD133作为肿瘤干细胞表面标志,采用流式细胞仪高速分选技术从人肺腺癌细胞株A549中分离肺癌干细胞及普通肿瘤细胞,实时荧光定量聚合酶链反应(FQ-PCR)及Western blot检测肺癌干细胞和普通肿瘤细胞内的Notch信号通路的表达水平;细胞计数试剂盒(CCK-8)检测肺癌干细胞和普通肿瘤细胞的体外增殖能力,并绘制生长曲线;使用γ-分泌酶抑制剂(DAPT)阻断Notch信号通路传导,观察肺癌干细胞和普通肿瘤细胞的体外生长差异.结果 流式分选前检测CD133阳性细胞(肺癌干细胞)占所有A549细胞的百分比为(0.40±0.11)％,而流式分选后所得细胞中,CD133阳性细胞占百分比为(95.00±0.63)％,两者差异有统计学意义(P＜0.01).Notch通路在肺癌干细胞及普通肿瘤细胞中均表达,Notch1及Notch2在肺癌干细胞中的表达显著低于在普通肿瘤细胞内的表达,差异有统计学意义(P ＜0.05);Hes1仅在普通肿瘤细胞中表达,在肺癌干细胞中未检测到表达.肺癌干细胞与普通肿瘤细胞的增殖能力差异无统计学意义(P＞0.05),而在DAPT干预48 h后,肺癌干细胞和普通肿瘤细胞的体外增殖均受到了抑制,肺癌干细胞的生长抑制率[(33.7±1.9)％]显著高于普通肿瘤细胞的生长抑制率[(21.5±3.4)％],差异有统计学意义(P＜0.05).结论 较之普通肿瘤细胞,阻断Notch通路传导对肺癌干细胞的生长抑制效果更强,这可能与DAPT抑制了Notch的过高表达对肺癌细胞的负反馈作用有关.     

激活的Notch信号系统对许旺细胞调控作用的影响

目的 研究体外激活的Notch信号系统对许旺细胞的调控作用.方法 培养成年SD大鼠坐骨神经许旺细胞,分别加入Notch信号激活剂Recombinant rat jagged1/FC chimera和抑制剂DAPT,空白对照加PBS缓冲液.通过免疫荧光检测细胞Notch信号蛋白激活状态,MTT检测细胞增殖情况,酶联免疫吸附试验检测细胞分泌NGF情况.结果 倒置显微镜下观察Notch激活组细胞生长优于其他各组;MTT检测该组促细胞增殖作用明显(P＜0.05),且增殖效果在72 h内逐渐增强;ELISA显示Notch激活组细胞培养上清液中NGF蛋白浓度[(60.11±2.61)pg/ml]较对照组[(51.29±3.59)pg/ml]及抑制组[(43.43±2.82 pg/ml]明显升高(P＜0.05).结论 一定剂量的Notch信号激活剂不仅促进体外培养的许旺细胞增殖,而且促进许旺细胞分泌NGF增多.     

Numb基因上调对人肾细胞癌细胞周期和增殖的影响

目的 研究Numb基因上调对人肾细胞癌的细胞周期和增殖能力的影响及相关机制.方法 选取人肾细胞癌细胞786-O为研究对象,使用Numb-ORF表达质粒转染细胞为实验组,设置阴性对照及空白对照组.采用荧光定量聚合酶链式反应和蛋白质印迹技术检测各组Numb基因和肿瘤增殖抗原Ki-67的表达;采用流式细胞技术检测细胞周期,计算G0/G1期细胞比例、增殖指数(PI)和S期分数(SPF);以MTS法检测细胞的增殖活力.结果 实验组Numb的相对表达水平为(18.97±1.49),显著的高于阴性对照组(3.34±0.41)和空白对照组(3.21±0.39);实验组Ki-67的相对表达水平为(4.31 ±0.58),明显少于阴性对照组(10.35±0.84)和空白对照组(9.89±0.73),差异有统计学意义(P＜0.01).细胞周期检测:实验组中G0/G1期细胞的比例(％)为60.47±1.58,明显高于阴性对照组(49.67±1.97)和空白对照组(52.52±2.47)(P＜0.01).实验组的PI和SPF均低于两对照组(P＜0.01).增殖检测:实验组在24、48、72h的吸光度(A)值均低于两对照组(P＜0.01).结论 在786-O细胞中上调Numb基因可以抑制Ki-67的表达,减低细胞的PI和SPF,促使细胞发生G0/G1期阻滞,抑制细胞的增殖活力,实验结果提示Numb基因在肾细胞癌中可能发挥抑癌因子作用.     

Notch信号通路在鱼藤酮诱导的PC12细胞凋亡中的作用

目的 探讨Notch信号通路在鱼藤酮诱导PC12细胞凋亡中的作用机制.方法 用鱼藤酮(5 μmol/L)处理PC12细胞,检测PC12细胞的凋亡和Notch信号通路的活化状态.同时以Notch信号抑制剂(2S)-N-N-(3,5-二氟苯乙酰基)-L-丙氨酰-2-苯基甘氨酸叔丁酯(DAPT,10 μmol/L)处理上述PC12细胞,观察Notch信号通路抑制后PC12细胞对鱼藤酮处理的凋亡反应,分析Notch信号通路与鱼藤酮诱导PC12细胞凋亡之间的关系.结果 鱼藤酮显著诱导PC12细胞凋亡[(35.6±5.4)％,P＜0.05]和半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)活性升高(0.52±0.15,P＜0.05),并活化其Notch/Jagged1信号通路.DAPT显著抑制鱼藤酮诱导的Notch/Jagged1信号通路活化并降低PC12细胞凋亡率[(9.8±3.1)％,P＜0.05]和Caspase-3活性(0.16 ±0.06,P＜0.05).结论 Notch信号通路活化是鱼藤酮诱导PC12细胞凋亡的主要机制之一.     

MicroRNA-126在肾透明细胞癌中的表达及对细胞株786-O生物学行为的影响

目的:研究MicroRNA-126(miRNA-126)在肾透明细胞癌(ccRCC)中的表达及其对细胞株786-O生物学行为的机制。方法:通过Real-time PCR的方法检测30例ccRCC及癌旁正常肾组织中miRNA-126-5p的表达情况。运用RT-qPCR的方法检测肾癌细胞株786-O和人肾小管上皮细胞株HK-2中miRNA-126-5p的表达;用Lipofectamine RNAiMAX将hsa-miRNA-126-5p转染786-O细胞株,用MTT、Transwell侵袭实验、Annexin V/PI双染法观察转染后细胞在增殖、侵袭力和凋亡方面的变化。结果:miRNA-126-5p在肾癌组织中的表达明显降低,在不同病理分级的样本中,miRNA-126-5p在高分化组中表达量为36.63±5.38,中、低分化组为19.96±7.28,差异无统计学意义(P0.05)。在不同临床分期的样本中,Ⅰ期miRNA-126-5p表达量为37.05±6.38,Ⅱ~Ⅲ期的表达量为24.03±6.15,不同分期之间差异无统计学意义(P0.05)。786-O细胞株转染后,检测miRNA-126-5p在实验组细胞的表达为42.86±7.01,显著高于阴性对照组27.46±8.43(P0.05)。MTT法检测786-O转染后48h增殖情况,实验组的细胞相对存活率为80.82±0.47,阴性对照组98.87±0.27,差异有统计学意义(P0.01)。Transwell侵袭实验显示,实验组miRNA-126-5p表达明显低于阴性对照组[(33.89±2.80)vs.(53.67±2.23),P0.05]。Annexin V/PI双染法检测细胞凋亡,实验组的细胞凋亡率明显高于阴性对照组[(22.56±1.13)vs.(7.20±0.26),P0.05]。结论:miRNA-126-5p在ccRCC组织中的表达量明显低于癌旁组织中的表达,其表达与肿瘤分级和临床分期无明显相关性。hsa-miRNA-126-5p转染细胞株786-O后,miRNA-126-5p的表达明显上调,并能够使786-O细胞增殖受到抑制,侵袭力降低,凋亡率增加。     

γ-分泌酶抑制剂3,5-二氟苯乙酰基-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯与氯喹的交叉对话作用对胃癌MKN-45细胞增殖、凋亡的影响

目的观察胃癌MKN-45细胞中Notch信号通路抑制剂3,5-二氟苯乙酰基-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)对自噬的影响及自噬抑制剂氯喹(CQ)对Notch信号通道的影响,探讨DAPT联合CQ对MKN-45细胞增殖、凋亡的影响。方法将MKN-45细胞分成4个组,对照组(Control组,RPMI 1640完全培养基培养);氯喹组(CQ组,含40μmol/L氯喹的RPMI 1640完全培养基培养);γ-分泌酶抑制剂组(DAPT组,含80μmol/L DAPT的RPMI 1640完全培养基培养);联合用药组(Comb组,含40μmol/L CQ和80μmol/L DAPT的RPMI 1640完全培养基培养)。应用噻唑蓝(MTT)法、流式细胞仪检测细胞增殖、凋亡;应用透射电镜、蛋白质印迹法(Western blot)、反转录-聚合酶链反应(RT-PCR)检测细胞自噬水平;应用Western blot检测Notch信号通路表达。两组间比较采用独立样本t检验。结果MTT结果显示,Comb组[(55.09±1.38)%]细胞增殖能力明显低于DAPT组[(78.83±1.74)%]和CQ组[(64.82±1.89)%],差异均有统计学意义(t=18.514、7.185,P<0.01)。流式细胞结果显示,Comb组[(43.43±2.30)%]凋亡率明显高于DAPT组[(27.26±3.02)%]和CQ组[(22.16±2.24)%],差异均有统计学意义(t=7.339、11.415,P<0.01)。Western blot、RT-PCR结果显示,DAPT组自噬相关的微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、苄氯素1(Beclin1)、自噬相关基因7(ATG7)信使核糖核酸(mRNA)、自噬相关基因12(ATG12)mRNA的相对表达量(0.587±0.030、1.503±0.066、1.797±0.110、2.433±0.111)明显高于Control组(0.333±0.002、1.137±0.068、1.000±0.000、1.000±0.000),差异均有统计学意义(t=14.893、6.695、12.527、22.446,P<0.01)。CQ组Notch1蛋白的相对表达量(0.932±0.061)明显高于Control组(0.477±0.031),差异有统计学意义(t=11.590,P<0.01)。结论胃癌MKN-45细胞中自噬与Notch信号通路存在交叉对话,两者相互调节,DAPT联合CQ可以使胃癌MKN-45细胞增殖能力下降、凋亡率提高。     

阻断Notch信号的角质形成细胞对成纤维细胞增殖及分泌功能的影响研究

目的:探讨在复合培养的条件下,将阻断Notch信号后的角质形成细胞与成纤维细胞共培养,对成纤维细胞增殖及表达角质细胞生长因子(Keratinocyte growth factor,KGF)、TGF-β1的影响。方法:运用角质形成细胞-成纤维细胞复合培养的模型,于复合培养前3d进行血清刺激或者γ-分泌酶抑制剂(DAPT)阻断角质形成细胞中的Notch信号,即在角质形成细胞分化前进行干预。然后提升至气液交界面使其达到复层生长和终末分化,后与成纤维细胞共培养,观察阻断Notch信号后的角质形成细胞对成纤维细胞增殖及分泌功能的影响。结果:分化前,给予角质形成细胞血清刺激,复合培养0h,Notch-1、Jagged-1表达明显升高(P0.05);复合培养第1天,P21表达上调、P63表达下降(P0.05)。血清刺激前给予DAPT预处理,复合培养0h,角质形成细胞中Notch信号下游分子P21和P63的表达恢复至无血清刺激水平(P0.05)。DAPT组的成纤维细胞增殖能力、KGF及TGF-β1的表达相对于血清刺激组明显被抑制(P0.05),而与无血清组比较,差异无统计学意义(P0.05)。结论:阻断Notch信号后的角质形成细胞与成纤维细胞共培养,能够明显抑制成纤维细胞的增殖及KGF、TGF-β1的合成。     

NNMT在肾透明细胞癌的表达及对肾癌细胞侵袭能力的影响

目的:研究NNMT在肾透明细胞癌中的表达情况及对肾癌细胞侵袭能力的影响。方法:采用RT-PCR和Western blot方法检测正常肾小管上皮细胞株HKC、肾癌细胞株786-O及30例肾透明细胞癌组织、相应癌旁组织中NNMT的mRNA和蛋白的表达水平,并分析NNMT的mRNA水平与临床病理参数的关系。化学合成针对NNMT特异的siRNA序列,应用脂质体Lipofectamine 2000将其转染进786-O细胞中,利用RT-PCR和Western blot法检测NNMT在786-O细胞中的表达水平,用Transwell小室法检测肾癌细胞786-O侵袭能力的变化。结果:NNMT在肾癌细胞786-O中的mRNA和蛋白表达水平显著高于正常肾小管上皮细胞株HKC(P<0.001);肾透明细胞癌组织和对应的癌旁组织中NNMT的mRNA相对表达量分别为(1.582±0.2145)、(0.1269±0.04279),两组比较P<0.001。NNMT的mRNA水平与肿瘤大小、临床分期有关(P<0.05);Tran-swell法检测结果显示降低NNMT的表达后786-O细胞的侵袭能力明显下降。结论:NNMT在肾透明细胞癌组织和细胞中表达升高,可能在肾癌发生、发展过程中发挥重要作用。     

shRNA靶向抑制CD74基因对人肾透明细胞癌细胞786-O生物学行为的影响

目的探讨抑制人CD74基因后对肾透明细胞癌786-O细胞生长和侵袭能力的影响。方法通过慢病毒lentivirus-CD74转染肾透明细胞癌786-O细胞后,MTT法检测786-O细胞活力的变化,利用流式细胞学检测肿瘤细胞的凋亡变化情况,Transwell小室实验检测786-O细胞侵袭能力改变情况,采用Westernblot方法检测转染前后786-O细胞CD74及相关蛋白的变化情况。结果慢病毒转染786-O细胞后细胞中CD74蛋白水平表达明显低于对照组(P0.05),抑制CD74蛋白表达水平后786-O细胞生长明显受到抑制,细胞活力降低,凋亡增加,细胞侵袭能力降低,且Survivin、MMP-2、MM-9蛋白也随之下降,其与对照组结果对比差异具有统计学差异(P0.05)。结论利用慢病毒转染技术抑制CD74的表达可以抑制肾透明细胞癌786-O细胞的增值和侵袭能力,CD74基因可能作为癌基因在肾透明细胞癌中起作用。     

金荞麦提取物调控DDIT4对肾癌细胞凋亡的影响

目的:探讨金荞麦提取物Fr4对肾癌细胞增殖和凋亡的影响及其作用机制。方法:采用不同浓度Fr4处理肾癌细胞786-O,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测786-O细胞中Cyclin D1、P21、Bcl-2、Bax、DDIT4表达。建立过表达DDIT4细胞。在786-O细胞中转染si-DDIT4,并用400μg/ml Fr4处理,研究786-O细胞增殖、凋亡变化。结果:Fr4明显抑制786-O细胞活性(P0.05),并呈剂量和时间依赖性;不同浓度Fr4显著减少Cyclin D1蛋白表达量,提高P21蛋白水平(P0.05),并呈剂量依赖性。400μg/ml的Fr4明显增加细胞凋亡率和Bax、DDIT4蛋白表达量(P0.05),降低Bcl-2蛋白水平。过表达DDIT4显著抑制24 h、48 h、72 h的细胞活性及Cyclin D1、Bcl-2蛋白水平,增加细胞凋亡率、P21和Bax蛋白表达量(P0.05)。抑制DDIT4逆转了Fr4对786-O细胞增殖、Cyclin D1、Bcl-2蛋白表达的抑制作用,和对细胞凋亡、DDIT4、P21和Bax蛋白表达的促进作用。结论:金荞麦提取物Fr4通过调控DDIT4表达抑制肾癌细胞增殖,并诱导其凋亡。     

Comparison of the Nellcor N-200 and N-3000 pulse oximeters during simulated postoperative activities

Twenty-six healthy volunteers were monitored simultaneously with the Nellcor N-200 and N-3000 pulse oximeters during nonhypoxaemic simulated postoperative activity. The overall number of registered events (hypoxaemic episodes or loss of signal) was fewer with the N-3000 than with the N-200 (8 vs. 32, p < 0.00005). Episodes of 'desaturation' of ≥5% from baseline were significantly fewer with the N-3000 than with the N-200 (5 vs. 19, p =0.0001), and lowest values below 90% occurred nine times on the N-200, but were not seen with the N-3000 (p <0.00005). Furthermore, episodes owing to loss of signal were significantly rarer with the N-3000 than with the N-200 (3 vs. 13, p =0.001). The Nellcor N-3000 oximeter may offer an advantage over the N-200 model when monitoring patients in the postoperative period.     

n一3多不饱和脂肪酸与骨代谢

骨质疏松是当今老龄社会的主要健康和经济问题,影响人类生活质量和寿命,基因因素、脂肪酸营养摄入等影响骨质疏松及其并发症。脂肪酸是细胞膜的重要组成部分,通过调节钙吸收、破骨细胞形成、前列腺素的合成、细胞膜n-6/n-3脂肪酸比率、细胞因子及脂质过氧化等可影响骨代谢。本文综述了膳食中n-3脂肪酸的重要性及fat-1基因对骨代谢的影响,n-3脂肪酸影响人类和鼠的骨代谢,分析n-3脂肪酸对骨代谢影响的细胞与分子机制。     

Continuous pulse oximetry in the general surgical ward: Nellcor N-200 versus Nellcor N-3000

New measurement principles in pulse oximetry have been introduced to decrease the incidence of false movement alarms. Experimental studies have shown that the new Nellcor Symphony N-3000 may reduce the incidence of false alarms when monitoring during different activities. We compared the Nellcor Symphony N-3000 with the Nellcor N-200 pulse oximeter, when monitoring patients in the general surgical ward. Twenty-two patients were monitored during unrestricted ward activities for a total of 275 h with a N-3000 and a N-200 pulse oximeter simultaneously. Data were analysed for lack of concordance between the two pulse oximeters with respect to frequency of registered hypoxaemic episodes and thus the amount of time spent in the alarm state. The median number of desaturation episodes with the N-200 was 18 (range 0-511) compared with four (range 0-476) with the N-3000 (p < 0.0001). The median number of drop-outs (loss of signal) was 13 (range 1-46) with the N-200 compared with nine (2-41) with the N-3000 (p = 0.06). The N-200 registered saturation values of 85% or below for 23% of the observation time compared with 6% of the observation time with the N-3000 pulse oximeter (p < 0.0001). The different working principles of the two generations of oximeters were reflected in the present results derived from a clinical setting. The Nellcor Symphony N-3000 may offer an advantage compared with the Nellcor N-200, because of the reduced frequency of alarms and total time in alarm when monitoring patients in the general surgical ward.     

The Nellcor N-101 pulse oximeter

The accuracy of the Nellcor N-101 pulse oximeter has been evaluated in adult patients receiving general anaesthesia or intensive care. Readings obtained noninvasively with this instrument were compared with measurements made on arterial blood using a Radiometer OSM2 oximeter. The pulse oximeter was easy to use and within the range tested (70-100 percent saturation of haemoglobin with oxygen) the readings were within I digit of the values obtained by in vitro measurement.     

Identification of N- and O-linked oligosaccharides in human seminal vesicles

The main oligosaccharide residues and the saccharide linkage in infantile and adult human seminal vesicles were studied by means of lectin histochemistry at light and electron microscopy levels. In adult glands, the epithelial cell cytoplasm and luminal content reacted positively to the following residues: (GlcNAc)n (WGA), Galbeta1,3GalNAc (PNA), GalNAcalpha1,3Gal (SBA), GalNAcalpha1,3GalNAc (HPA), Fucalpha1,2Galbeta1,4GlcNAc (UEA-I), and alphaL-Fuc1,6DGlcNAc-O-Melibiosc (AAA). The presence of intense staining in the luminal content suggest that glycoproteins containing these oligosaccharide moieties are secreted by epithelial cells. Adult epithelial cells also reacted to Neu5Acalpha2,6Gal (SNA), Neu5Acalphaa2,3Galbeta1,4GlcNAc (MAA), Galbeta1,4GlcNAc (DSA), branched mannose chains (ConA), Man1,3Man (GNA), and Fucalpha1,2Galbeta1,4GlcNAcFucalpha1,3GlcNAc (LTA) but reaction to these residues was weak (MAA, DSA, ConA, and LTA) or absent (SNA and GNA) in the gland lumen, which suggests that they belong to intracytoplasmic proteins. The chemical and enzymatic treatments used suggest that the residues recognized by SNA, MAA, PNA, DSA, HPA, and SBA belong to O-linked oligosaccharides; those residues localized by ConA and GNA have an N-glycosidic linkage, and those bound by WGA, LTA, UEA-I, and AAA are linked to both N- and O-oligosaccharides. In prepubertal seminal vesicles, reaction in the epithelial cell cytoplasm was similar to that observed in adults, except for GNA and HPA, which showed a weaker reaction. However, the lumen of prepubertal seminal vesicles showed intense reaction to WGA and SBA only. The chemical and enzymatic treatments suggest that the scanty glycoproteins secreted by the prepubertal glands belong to the mucin-type.     

Expression and localization of N- and E-cadherin in the human testis and epididymis

Cellular interactions in the testis and epididymis are an important prerequisite for spermatogenesis and sperm maturation, and involve a well-developed complex of intercellular junctions. Cadherins are cell surface proteins which mediate intercellular Ca2+ -dependent adhesion and are believed to be fundamentally important for maintaining multicellular structures. In the present study we report the expression of a 135 kDa N-cadherin polypeptide in the human seminiferous epithelium by immunoblotting. The presence of N-cadherin was demonstrated by immunohistochemistry on the surface of spermatogonia and primary spermatocytes, and possibly also around some early spermatids, whereas late spermatids were always negative. Endothelial cells also stained for N-cadherin, whereas peritubular cells and Leydig cells did not. No expression of E-cadherin could be demonstrated in the human testis. In the human epididymis E-cadherin, but not N-cadherin, was expressed and localized to the surface of the principal epithelial cells as shown by immunohistochemistry. These observations indicate that cadherins play an important role in the organization of the seminiferous and epididymal epithelium.     

Distribution of N- and O-linked oligosaccharides on surface of spermatozoa from normal and infertile subjects

Sperm glycocalyx modifications are very important for gamete recognition and fertilisation in mammals. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. The purpose of this study was to determine the distribution of surface carbohydrates in human spermatozoa from normal and oligospermic subjects. Fifteen ejaculates each from normal fertile and oligospermic individuals were analysed. N-linked and O-linked surface carbohydrates were detected by fluorescence microscopy using fluorescein isothiocynate-conjugated lectins. Triticum vulgaris agglutinin (WGA)-binding sites were found to be decreased on acrosomal domain in spermatozoa from oligospermic individuals, while no changes were observed in the binding sites of Concanavalin ensiformis, Peanut agglutinin and Lens clunaris agglutinin. A reduction in binding sites for soybean agglutinin and Ricinus communis agglutinin was observed on the acrosomal domains in spermatozoa from oligospermic individuals. Changes in sperm glycocalyx observed in this study provide new insights into molecular rearrangement of sperm membrane in infertility.     

Influences of the N- and C-termini of the distal nephron inward rectifier, ROMK

The inward rectifying potassium channels of the ROMK family are present in the distal nephron of the kidney. These channels have two membrane spanning portions, between which lies a hydrophobic domain thought to confer the majority of the conductive properties of the channel. The N- and C-termini are both intracellular. In this paper we have examined the contribution of the N- and C-termini to the pore by examining the interaction of Cs+ with the channels. ROMK1 has an additional 19 amino acids on its N-terminus in comparison to ROMK2. The C-terminus of ROMK2 was extended by addition of a streptavidin tag (sfROMK2). Currents were measured following expression in Xenopus oocytes using two-electrode voltage clamp. ROMK1, ROMK2 and sfROMK2 exhibited concentration- and voltage-dependent block of inward currents by extracellular Cs+. The Hill coefficients were not significantly different from one. The mean Kd values at 0 mV were 100.6 +/- 10.6, 63.1 +/- 3.9 and 40.6 +/- 9.4, respectively (p < 0.05). The electric distances (delta) were 0.94 +/- 0.06, 1.0 +/- 0.05 and 1.37 +/- 0.06 respectively. The delta of sfROMK2 was greater than either ROMK1 or ROMK2 (p < 0.001). ROMK1, ROMK2 and sfROMK2 are sensitive to extracellular Cs+. Block was both concentration- and voltage-dependent. sfROMK2 is most Cs+-sensitive. ROMK1 contains an additional N-terminal 19 amino acids. Thus the pore properties of these two isoforms are subtly different, and influenced by the N-terminus. The lower Kd in sfROMK2 suggests that the streptavidin tag, and perhaps the C-terminus, also affect the pore.