碘化N-正丁基氟哌啶醇对豚鼠心房肌细胞乙酰胆碱敏感性钾通道的影响

目的研究碘化N正丁基氟哌啶醇(F2)对豚鼠心房肌细胞乙酰胆碱敏感性钾通道(KACh)的影响,探讨其对KACh的作用机制。方法采用膜片钳全细胞记录方法,测定F2对原代培养的豚鼠心房肌细胞乙酰胆碱敏感性钾电流IK(ACh)的影响。结果细胞外给予F2对豚鼠心房肌细胞IK(ACh)呈可逆性、浓度依赖性的阻断作用。细胞内添入抗水解的GTP类似物GTPγS后,结果同前。细胞内给予50μmol·L-1F2对IK(ACh)无作用。结论F2是豚鼠心房肌细胞KACh的一种快速通道阻断剂,发挥作用部位在细胞膜外侧,作用位点在钾通道本身,与乙酰胆碱受体无关。     

地高辛衍生物对人胚肾细胞293延迟整流钾电流的作用

目的 研究地高辛衍生物对人胚肾细胞(human embryonic kidney 293 cell,HEK-293)上延迟整流钾电流(delayed after potassium current,IK)的影响.方法 应用磷酸钙瞬时转染的方法,将人心肌细胞上的延迟整流钾电流通道蛋白基因(human Ether-a-go-go Related Gene,HERG)转染进人胚肾细胞,全细胞膜片钳技术记录药物浓度分别为1、10、100 nmol·L-1的地高辛甲、乙两种衍生物对人胚肾细胞上HERG钾通道时间依赖性电流(Istep)和尾电流(I tail)的影响.结果 地高辛衍生物甲和地高辛类似,对HERG钾通道时间依赖性电流和尾电流均呈现剂量依赖性抑制,其半数最大抑制浓度IC50分别为20.61、22.69 nmol·L-1;未见地高辛衍生物乙对HERG钾通道延迟整流钾电流的抑制作用.结论 地高辛衍生物甲对转染HERG钾通道的人胚肾细胞的延迟整流钾电流呈浓度依赖性抑制;而地高辛衍生物乙对转染HERG钾通道的人胚肾细胞的延迟整流钾电流无抑制作用.     

延胡索乙素对豚鼠单个心室肌细胞钾离子通道的影响

目的研究延胡索乙素(dltetrahydropalmatine,THP)对正常豚鼠心室肌细胞钾电流的影响,旨在探讨延胡索乙素抗心律失常作用机制。方法酶解法分离豚鼠单个心室肌细胞,应用全细胞膜片钳技术记录延胡索乙素对豚鼠单个心室肌细胞钾电流的影响。结果延胡索乙素可明显抑制延迟整流钾电流(IK)和内向整流钾电流(IK1),并呈剂量依赖性。结论延胡索乙素抗心律失常作用机制可能与它对心肌细胞钾通道作用有关,THP可抑制IK和IK1,使动作电位时程(APD)和有效不应期(ERP)延长从而发挥其抗心律失常作用。     

卡维地洛对HERG钾通道电生理功能及蛋白表达的影响

目的探讨卡维地洛对稳定转染HERG基因的HEK(human embryonic kidney)293细胞上的HERG钾通道的影响。方法应用全细胞膜片钳技术记录在HEK293细胞上稳定表达的HERG钾通道的电流和动力学曲线(激活、失活、复活和去激活),研究不同浓度卡维地洛对HERG电流及动力学的影响;用Western blot技术定量的观察不同浓度卡维地洛对定位于HERG-HEK细胞膜上的HERG钾通道蛋白表达的影响。结果卡维地洛(10、100nmol.L-1,1、10μmol.L-1)浓度依赖性的抑制HERG步阶电流(Istep)及其尾电流(Itail)。由Hill方程得出IC50为539.6nmol.L-1,Hillslope为-0.64。1μmol.L-1卡维地洛作用后瞬时失活、去激活时间常数明显降低(P<0.05,n=10),而激活、失活、复活动力学无明显改变;Western blot检测结果显示卡维地洛组与无加药对照组HERG蛋白的表达无差别。结论卡维地洛通过影响通道的开放状态抑制HERG钾电流,加速瞬时失活和去激活动力学,对HERG蛋白的表达及成熟无影响。     

“非抗菌药物剂量和频次智能化监控系统”的研发

目的研究3,5,4'-三甲基白藜芦醇(trans-resveratrol derivative3,5,4'-trimethoxystilbene,TMS)对豚鼠心室肌细胞钠电流(INa)和钾电流(IK1)的直接作用,探讨其心肌保护作用。方法用全细胞膜片钳技术记录TMS对单个心室肌细胞INa和IK1的作用。结果TMS(10μmol·L-1)可快速抑制豚鼠心室肌细胞INa,用药后3min左右即开始起效,10min时抑制率为(36.8±5.6)%(P<0.005),洗脱后可完全恢复;1,3μmol·L-1TMS未影响INa大小。TMS不改变INa的最大激活电压,也不影响IK1的大小。10μmol·L-1使半数最大失活电压(V1/2)由(-87.0±3.3)mV变化到(-96.7±3.5)mV(P<0.001),使失活曲线斜率(S)由(4.9±0.3)mV变化到(5.4±0.3)mV(P<0.01);使半数最大激活电压(V1/2)(-38.9±1.4)mV变化到(-47.3±1.3)mV(P<0.001),未改变激活S。结论TMS可直接作用于豚鼠心室肌细胞,快速抑制INa,且此作用快速、可逆。     

小檗碱对心肌细胞I_(K1)、I_K及HERG通道的抑制作用(英文)

目的:研究小檗碱(Ber)对豚鼠心室肌细胞钾通道和动作电位作用,以及在蛙卵中表达的人的HERG通道的作用。方法:酶解方法分离单个心肌细胞,采用全细胞膜片箝方法记录钾离子电流及动作电位,基因箝技术研究HERG通道电流。结果:Ber可显著延长动作电位时程,并呈剂量依赖性。Ber 100μmol/L使APD_(90)由对照的(450±48)ms延长至(888±90)ms(n=6,P<0.01)。Ber对I_(Kl)及I_K呈剂量依赖性抑制作用。Ber 100μmol/L对I_(Kl)的抑制率达65％±7％(n=6,P<0.01)。Ber 50μmol/L对I_K的抑制率达57％±6％;对I_(Ktail)的抑制率达53％±6％。Ber对I_K作用呈现电压依赖性。Ber对在蛙卵中表达的HERG通道具有很强的阻断作用,IC_(50)为75μmol/L,此阻断作用也呈电压依赖性。结论:Ber可使动作电位时程明显延长,对I_(Kl)及I_K具有阻断作用。Ber可显著抑制HERG通道。Ber抗心律失常的机制与其抑制I_(Kl)、I_K及HERG通道密切相关。     

甲基苯丙胺对豚鼠心室肌细胞动作电位及延迟整流钾电流的影响(英文)

目的研究甲基苯丙胺对心血管系统的毒性作用及其机制。方法分离豚鼠心室肌细胞,采用全细胞膜片钳技术获取并分析心室肌细胞延迟整流钾电流(IK)及动作电位(AP)水平。结果甲基苯丙胺0.5mmol·L-1使豚鼠心室肌细胞AP幅值从121.6mV降至106.0mV,能延长动作电位时程(APD),但不改变静息电位水平。其中动作电位复极10%,25%,50%,75%及90%时程(APD10,APD25,APD50,APD75,APD90)分别延长179.0%,88.7%,47.7%,43.4%和31.9%(P<0.05)。甲基苯丙胺0.5mmol·L-1使快速激活延迟整流钾电流(IKr)和缓慢激活延迟整流钾电流(IKs)的膜电位水平降低,电流-电压曲线下移,但曲线形状不变,冲洗后能部分恢复。用含甲基苯丙胺0.01,0.1,0.5,1.0和3.0mmol·L-1的细胞外液分别灌流细胞5min,甲基苯丙胺对IKr尾电流幅度呈浓度依赖性的阻断作用,冲洗后能部分恢复。甲基苯丙胺对IKs尾电流的影响也非常显著(P<0.05)。结论甲基苯丙胺对豚鼠心室肌细胞的IK及AP都有不同程度的影响,这可能是甲基苯丙胺造成心脏损伤的电生理机制之一。     

白藜芦醇对HERG钾通道电生理功能的影响

目的探讨白藜芦醇对稳定转染HERG基因的HEK293细胞上HERG钾通道的影响,进一步了解其抗心律失常的作用机制。方法传代得到单个HERG-HEK细胞,应用全细胞膜片钳技术记录HERG-HEK细胞上的HERG钾电流和动力学曲线(激活、失活、复活和去活化)。结果白藜芦醇(1、10、100μmol·L-1)浓度依赖性的抑制HERG步阶电流(IHERG)及其尾电流(IHERGtail),0mV电压下1μmol·L-1白藜芦醇抑制IHERG25.3%±9.4%,IHERGtail23.6%±11%;10μmol·L-1白藜芦醇抑制IHERG28%±8.4%,IHERGtail28.8%±9.1%;100μmol.L-1白藜芦醇抑制IHERG43.7%±1.5%,IHERGtail47.9%±11%(P<0.05,n=12)。100μmol·L-1白藜芦醇作用后失活时间常数减小,失活速率变快;去活化时间常数明显减小(P<0.05,n=12)。激活、瞬时失活和复活动力学没有明显变化。结论白藜芦醇通过影响通道的开放状态和失活状态抑制HERG钾电流,从而使得心肌细胞复极时间延长,改善快速性心律失常。     

牛磺酸镁对豚鼠心室肌细胞钾离子通道的影响

目的研究牛磺酸镁(taurine magnesium coordination compound,TMC)对正常豚鼠心室肌细胞钾电流的影响,旨在探讨TMC抗心律失常的作用机制。方法酶解法分离豚鼠单个心室肌细胞,应用全细胞膜片钳技术记录豚鼠单个心室肌细胞IK、IK1的影响。结果应用200μmol·L-1TMC使豚鼠单个心室肌细胞IK在实验电压+70mV时,从给药前(8.67±1.04)pA/pF减少到(6.31±1.16)pA/pF(n=5,P<0.01);TMC对IK1无影响。结论本实验表明TMC具有直接抑制心室肌细胞IK作用,减少IK可能会使动作电位时程(APD)和有效不应期(ERP)延长,这可能是其发挥抗心律失常作用的基础之一。     

葡萄糖对豚鼠心室肌细胞跨膜离子电流的影响

目的观察葡萄糖对豚鼠心室肌细胞膜APD,IK1,IK,ICa-L的影响.方法采用全细胞膜片技术记录单个豚鼠心室肌细胞膜的动作电位时程和离子电流.比较0、10和20mmol·L-1葡萄糖对心室肌细胞跨膜离子电流的作用.结果(1)与10 mmol·L-1葡萄糖相比,0和20mmol·L-1均可使豚鼠心室肌细胞的APD缩短(P＜0.05);(2)在细胞外葡萄糖浓度为10 mmol·L-1时,内向整流钾电流IK1的电流密度最大,标准化I-V曲线显示0和20mmol·L-1葡萄糖均可抑制IK1,并使I-V曲线左移,其翻转电位从-72.4移至-64.6 mV;(3)与mmol·L-1葡萄糖相比,0和20mmol·L-1葡萄糖均可增加ICa-L的电流幅度和电流密度.当钳制电位为10 mV,细胞外葡萄糖浓度为0 mmol·L-1时,ICa-L的电流密度为(-8.03±0.82)pA/pF(n=8),而10mmol·L-1和20mmol·L-1葡萄糖分别使电流密度增加为(-5.45±0.67)pA/pF和(-6.50±0.56)pA/pF;(4)当钳制电压为 70mV,细胞外葡萄糖浓度为0、10和20 mmol·L-1时,IK的电流密度分别为(18.96±2.86)pA/pF,(8.66±1.87)pA/pF,(15.32±3.12)pA/pF.结论细胞外不同浓度的葡萄糖可使豚鼠心室肌细胞APD,IK1,IK,ICa-L发生改变.细胞外葡萄糖浓度为0和20mmol·L-1对细胞膜离子电流产生相似的影响.     

Blockade of HERG K+ channel by an antihistamine drug brompheniramine requires the channel binding within the S6 residue Y652 and F656

A number of clinically used drugs block delayed rectifier K+ channels and prolong the duration of cardiac action potentials associated with long QT syndrome. This study investigated the molecular mechanisms of voltage-dependent inhibition of human ether-a-go-go-related gene (HERG) delayed rectifier K+ channels expressed in HEK-293 cells by brompheniramine, an antihistamine. Brompheniramine inhibited HERG current in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) value of 1.7 microm at 0 mV. A block of HERG current by brompheniramine was enhanced by progressive membrane depolarization and showed significantly negative shift in voltage-dependence of channel activation. Inhibition of HERG current by brompheniramine showed time-dependence. The S6 residue HERG mutant Y652A and F656C largely reduced the blocking potency of HERG current. These results indicate that brompheniramine mainly inhibited the HERG potassium channel through the residue Y652 and F656 and these residues may be an obligatory determinant in inhibition of HERG current for brompheniramine.     

Fluconazole inhibits hERG K(+) channel by direct block and disruption of protein trafficking

Fluconazole, a commonly used azole antifungal drug, can induce QT prolongation, which may lead to Torsades de Pointes and sudden death. To investigate the arrhythmogenic side effects of fluconazole, we studied the effect of fluconazole on human ether-a-go-go-related gene (hERG) K⁺ channels (wild type, Y652A and F656C) expressed in human embryonic kidney (HEK293) cells using a whole-cell patch clamp technique, Western blot analysis and confocal microscopy. Fluconazole inhibited wild type hERG currents in a concentration-dependent manner, with a half-maximum block concentration (IC₅₀) of 48.2 ± 9.4 μM. Fluconazole did not change other channel kinetics (activation and steady-state inactivation) of hERG channel. Mutations in drug- binding sites (Y652A or F656C) of the hERG channel significantly attenuated the hERG current blockade by fluconazole. In addition, fluconazole inhibited the trafficking of hERG protein by Western blot analysis and confocal microscopy, respectively. These findings indicate that fluconazole may cause acquired long QT syndrome (LQTS) via a direct inhibition of hERG current and by disrupting hERG protein trafficking, and the mutations Y652 and F656 may be obligatory determinants in inhibition of hERG current for fluconazole.     

Inhibition of cardiac HERG potassium channels by antidepressant maprotiline

Many drugs block delayed rectifier K+ channels and prolong the cardiac action potential duration. Here we investigate the molecular mechanisms of voltage-dependent block of human ether-a-go-go-related gene (HERG) K+ channels expressed in cells HEK-293 and Xenopus oocytes by maprotiline. The IC50 determined at 0 mV on HERG expressed HEK-293 cell and oocytes was 5.2 and 23.7 microM, respectively. Block of HERG expressed in oocytes by maprotiline was enhanced by progressive membrane depolarization and accompanied by a negative shift in the voltage dependence of channel activation. The potency of maprotiline was reduced 7-fold by point mutation of a key aromatic residue (F656T) and 3-fold for Y652A, both located in the S6 domain. The mutation Y652A inverted the voltage dependence of HERG channel block by maprotiline. Together, these results suggest that voltage-dependent block of HERG results from gating dependent changes in the accessibility of Y652, a critical component of the drug binding site.     

Comparison of HERG channel blocking effects of various beta-blockers-- implication for clinical strategy

beta-Blockers are widely used in the treatment of cardiovascular diseases. However, their effects on HERG channels at comparable conditions remain to be defined. We investigated the direct acute effects of beta-blockers on HERG current and the molecular basis of drug binding to HERG channels with mutations of putative common binding site (Y652A and F656C). beta-Blockers were selected based on the receptor subtype. Wild-type, Y652A and F656C mutants of HERG channel were stably expressed in HEK293 cells, and the current was recorded by using whole-cell patch-clamp technique (23 degrees C). Carvedilol (nonselective), propranolol (nonselective) and ICI 118551 (beta(2)-selective) inhibited HERG current in a concentration-dependent manner (IC(50) 0.51, 3.9 and 9.2 microM, respectively). The IC(50) value for carvedilol was a clinically relevant concentration. High metoprolol (beta(1)-selective) concentrations were required for blockade (IC(50) 145 microM), and atenolol (beta(1)-selective) did not inhibit the HERG current.Inhibition of HERG current by carvedilol, propranolol and ICI 118551 was partially but significantly attenuated in Y652A and F656C mutant channels. Affinities of metoprolol to Y652A and F656C mutant channels were not different compared with the wild-type. HERG current block by all beta-blockers was not frequency-dependent. Drug affinities to HERG channels were different in beta-blockers. Our results provide additional strategies for clinical usage of beta-blockers. Atenolol and metoprolol may be preferable for patients with type 1 and 2 long QT syndrome. Carvedilol has a class III antiarrhythmic effect, which may provide the rationale for a favourable clinical outcome compared with other beta-blockers as suggested in the recent COMET (Carvedilol Or Metoprolol European Trial) substudy.     

Inhibition of cardiac HERG currents by the DNA topoisomerase II inhibitor amsacrine: mode of action

1 The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether-a-go-go-related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. 2 HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch-clamp and two-microelectrode voltage-clamp electrophysiology. 3 Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner, with IC50 values of 209.4 nm and 2.0 microm, respectively. 4 HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (-7.6 mV) and inactivation (-7.6 mV). HERG current block by amsacrine was not frequency dependent. 5 The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore-S6 region. 6 In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.     

Verapamil blocks HERG channel by the helix residue Y652 and F656 in the S6 transmembrane domain

Aim： The objectives of this study were to investigate the inhibitory action of verapamil on wild-type（WT） and mutation HERG K^＋ channel current （IHERG）, and to determine whether mutations in the S6 region ale important for the inhibition of IHERG by verapamil. Methods： HERG channels （WT, Y652A, and F656A） were expressed in oocytes of Xenopus laevis and studied using the 2-electrode voltage-clamp technique. Results： WT HERG is blocked in a concentration-dependent manner by verapamil （half-maximal inhibition concentration [IC50]=5.1 μmol/L）, and the steady state activation and inactivation parameters are shifted to more negative values. However, mutation to Ala of Y652 and F656 located on the S6 domain produced 16-fold and 20-fold increases in IC50 for IHERG blockade, respectively. Simultaneously, the steady state activation and inactivation parameters for Y652A are also shifted to more negative values in the presence of the blockers. Conclusion： Verapamil preferentially binds to and blocks open HERG channels. Tyr-652 and Phe-656, 2 aromatic amino-acid residues in the inner （S6） helix, are critical in the verapamil-binding site.     

Block of hERG channel by ziprasidone: biophysical properties and molecular determinants

Ziprasidone, an antipsychotic agent, delays cardiac repolarization and, thus, prolongs the QT interval of the cardiac ECG. In this study, we examined the biophysical properties and the molecular determinants of the ziprasidone block of wild-type hERG potassium channels stably expressed in HEK-293 cells or wild-type and mutant hERG channels expressed in Xenopus oocytes. In stably transfected HEK-293 cells, ziprasidone blocked wild-type hERG current in a voltage- and concentration-dependent manner (IC(50)=120nM, 0mV, 37 degrees C). Ziprasidone showed minimal tonic block of hERG current estimated during a depolarizing voltage (-20 or +30mV) or evaluated by the envelope of tails test (+30mV). Rate of the block onset was rapid, but not significantly affected by test potentials ranging from -20 to +30mV (time constant (tau)=114+/-14ms at +30mV). The time constant of the slow component of hERG current deactivation (at -50mV) was significantly increased by ziprasidone (tau=1776+/-90 versus 1008+/-71ms, P<0.01). Time course of channel inactivation was slowed by ziprasidone in a voltage-dependent manner. The V(1/2) values for steady-state activation and inactivation of hERG channel in HEK-293 cells were not significantly altered by ziprasidone. In Xenopus oocytes, ziprasidone exhibited less potent block of wild-type hERG current (IC(50)=2.8microM, 0mV, 23 degrees C). Mutation of the aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of hERG dramatically reduced the potency of channel block by ziprasidone (IC(50)>0.4 and 1mM at 0mV for Y652A and F656A, respectively). In conclusion, ziprasidone preferentially binds to and blocks open hERG channels. Tyr-652 and Phe-656 are two critical residues in the ziprasidone-binding site.     

HEK293细胞—— 一种研究受体Ca~(2+)调控功能的理想模型

目的了解ＨＥＫ２９３细胞Ｃａ２＋代谢的生物学特性。方法用Ｆｕｒａ－２荧光探针双波长测定细胞胞浆游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）方法，观察多种能改变细胞内Ｃａ２＋代谢的药物对天然的和转染了α１Ｂ肾上腺素受体ｃＤＮＡ的ＨＥＫ２９３细胞［Ｃａ２＋］ｉ的影响。结果在含１．５ｍｍｏｌＬ－１ＣａＣｌ２的缓冲液中，ＫＣｌ５０ｍｍｏｌＬ－１和ＢａｙＫ８６４４１０μｍｏｌＬ－１不影响ＨＥＫ２９３细胞的［Ｃａ２＋］ｉ；ｃｙｃｌｏｐｉａｚｏｎｉｃａｃｉｄ（ＣＰＡ０．０１，０．１，１０μｍｏｌＬ－１）能浓度依赖性地引起ＨＥＫ２９３细胞的［Ｃａ２＋］ｉ呈双相升高，其中的Ｃａ２＋内流相不受ｎｉｆｅｄｉｐｉｎｅ（１０μｍｏｌＬ－１）的影响；但可被１ｍｍｏｌＬ－１ＮｉＳＯ４完全抑制。在无Ｃａ２＋的缓冲液中，咖啡因２０ｍｍｏｌＬ－１和ｒｙａｎｏｄｉｎｅ１μｍｏｌＬ－１均不影响ＨＥＫ２９３细胞的［Ｃａ２＋］ｉ。先用ＣＰＡ（３０μｍｏｌＬ－１）耗竭ＨＥＫα１Ｂ细胞内Ｃａ２＋贮存池后，肾上腺素１０μｍｏｌＬ－１能进一步升高［Ｃａ２＋］ｉ。在有Ｃａ２＋或无Ｃａ２＋的缓冲液中，肾上腺素均可引起ＨＥＫα１Ｂ细胞［Ｃａ２＋］ｉ升高。结论ＨＥＫ２９３细胞?     

Intracellular K+ is required for the inactivation-induced high-affinity binding of cisapride to HERG channels

Many commonly used medications can cause long QT syndrome and thus increase the risk of life-threatening arrhythmias. High-affinity human Ether-à-go-go-related gene (HERG) potassium channel blockade by structurally diverse compounds is almost exclusively responsible for this side effect. Understanding drug-HERG channel interactions is an important step in avoiding drug-induced long QT syndromes. Previous studies have found that disrupting HERG inactivation reduces the degree of drug block and have suggested that the inactivated state is the preferential state for drug binding to HERG channels. However, recent studies have also shown that inactivation does not dictate drug sensitivity of HERG channels. In the present study, we examined the effect of inactivation gating on cisapride block of HERG. Modulation of HERG inactivation was achieved by either changing extracellular K+ or Cs+ concentrations or by mutations of the channel. We found that although inactivation facilitated cisapride block of the HERG K+ current, it was not coupled with cisapride block of HERG when the Cs+ current was recorded. Furthermore, cisapride block of the HERG K+ current was not linked with inactivation in the mutant HERG channels F656V and F656M. Our results suggest that inactivation facilitates cisapride block of HERG channels through affecting the positioning of Phe-656.