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The human ether-a-go-go related gene (HERG) encodes the α -subunit of the rapid component of the delayed rectifier K(+) channel, which is essential for the third repolarization of the action potential of human myocardial cells. Mutations of the HERG gene can cause type II hereditary long QT syndrome (LQT2), characterized by prolongation of the QT interval, abnormal T wave, torsade de pointes, syncope and sudden cardiac death. So far more than 300 HERG mutations have been identified, the majority of which can cause LQT2 due to HERG protein trafficking defect. It has been reported that certain drugs can induce acquired long QT syndrome through directly blocking the pore and/or affecting the HERG trafficking. The trafficking defects and K(+) currents can be restored with low temperature and certain drugs. However, the mechanisms underlying defective trafficking caused by HERG mutations and the inhibition/restoration of HERG trafficking by drugs are still unknown. This review summarizes the current understanding of the molecular mechanisms including HERG trafficking under physiological and pathological conditions, and the effects of drugs on the HERG trafficking, in order to provide theoretical evidence for the diagnosis and treatment of long QT syndrome.

Objective: To construct the pcDNA3-HERG-G572R expression vector and establish a cell line stably expressing HKE-HERG-G572R., Methods: HERG-G572R mutant fragment was constructed by over-lap extension PCR and validated by DNA sequencing. The HKE-HERG-G572R expression vector was constructed and transfected into HEK293 cells to obtain a cell line stably expressing HKE-HERG-G572R., Results: The pcDNA3-HERG-G572R expression vector was successfully constructed and the cell line stably expressing HKE-HERG-G572R was established. Real-time PCR and Western blotting revealed a 632-fold HKE-HERG-G572R overexpression in the transfected HEK293 cells as compared with that in control HEK293 cells transfected with pcDNA3 (P<0.01)., Conclusion: The protocol can be used to construct the cell line stably expressing HKE-HERG-G572R to provide a cell model for studying individualized therapy.

Objective: To investigate KCNQ1, KCNH2, KCNE1 and KCNE2 gene variants in the cases of sudden manhood death syndrome (SMDS)., Methods: One hundred and sixteen sporadic cases of SMDS and one hundred and twenty-five healthy controlled samples were enrolled. Genomic DNA was extracted from blood samples. Gene variants of KCNQ1, KCNH2, KCNE1 and KCNE2 were screened by direct sequencing., Results: A total of 14 mutations and 14 SNP were detected. Two non-synonymous mutations of them were newfound. There was no non-synonymous mutation found in the control group., Conclusion: There are KCNQ1, KCNH2, KCNE1 and KCNE2 gene variants found in Chinese SMDS cases. KCNQ1, KCNH2, KCNE1 and KCNE2 gene mutation may correlate partly with the occurrence of some cases of the SMDS in China.

Objective: To perform mutation analysis in a family with long QT syndrome., Methods: The medical record of the affected child and his parents were collected. The locus of gene associated with the long QT syndrome was mapped by linkage analysis. Mutation analysis was done by PCR-single strand conformation polymorphism (SSCP) and direct sequencing., Results: A mutation (L539fs/47) and a SNP (L564L) were found in exon 7 of the KCNH2 gene of the proband. The mutation was from the father., Conclusion: A novel mutation of L539fs/47 in the KCNH2 gene was identified in the LQTS family, which might be the disease-causing mutation for the family.

Objective: The congenital long QT syndrome (LQTs) is a hereditary disorder in which most affected family members have delayed ventricular repolarization manifested on the electrocardiogram (ECG) as QT interval prolongation. The disorder is associated with an increased propensity to arrhythmogenic syncope, polymorphous ventricular tachycardia (torsade de pointes), and sudden arrhythmic death. LQTs is due to mutations involving principally the myocyte ion-channels, and this monogenetic disorder has an autosomal inheritance pattern. This study investigated the gene mutation of a Chinese family of LQTs with multiple phenotypes including dilated cardiomyopathy (DCM) and cardiac conduction defects, thus to understand the molecular pathogenesis of the diseases., Methods: A three-generation Chinese LQTs family with multiple phenotypes was investigated. Blood sample was collected from the 8 family members and 100 unassociated normal individuals. Polymerase chain reaction (PCR)-DNA direct sequencing was performed to screen all exons and their flanking introns of SCN5A gene for mutation analysis. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to exclude polymorphism., Results: PCR amplification and subsequent direct sequencing of SCN5A from proband revealed a heterozygous deletion of nine base pairs (CAGAAGCCC) in exon 26, corresponding to the three amino acid residues Gln1507-Lys1508-Pro1509 (QKP). This mutation is localized in the linker region between DIII-DIV of SCN5A. The same mutation was found in another patient (her grandmother) and excluded in the remaining living subjects in this family. This mutation was confirmed using SSCP in 100 unassociated healthy individuals. Similar analysis excluded possible mutations that would lead to amino acid changes in KCNQ1, KCNH2 and LAMIN A/C commonly associated with LQTs and DCM with conduction disorders, no new mutations that would lead to amino acid changes was found., Conclusion: The result of the present study suggests that SCN5A mutation delQKP1507-1509 exists in patients with LQTs. The delQKP1507-1509 of SCN5A is a novel mutation in Chinese people. The same mutation was previously reported in a French family with only a single LQTs phenotype. Further studies on functional expression of SCN5A mutation delQKP1507-1509 will be helpful to understand the mechanism of the multiple phenotypes.

Objective: To explore the effects of eukaryotic expression vector pcDNA3-HERG transfection on angiotensin II (Ang II) induced myocyte hypertrophy in cultured neonatal rabbit ventricular myocytes., Methods: Neonatal rabbit ventricular myocytes and eukaryotic expression vector pcDNA3-HERG transfected ventricular myocytes were cultured in Dulbecco's-modified Eagle medium (DMEM), containing 1% fetal bovine serum (FBS) for 6 h, then stimulated with Ang II (10(-7) mol/L) for 48 h. Control ventricular myocytes were cultured in Dulbecco's-modified Eagle medium (DMEM), containing 1% fetal bovine serum (FBS) for 54 h. At 6 and 54 h, myocyte hypertrophic parameters including myocyte volume, total protein content and membrane capacitance, action potential duration (APD) and Calcineurin (CaN) activity were measured., Results: Compared to control myocytes, APD at 90% repolarization (APD(90)) was prolonged by 19.8% (P < 0.01), without signs of myocyte hypertrophy at 6 h post Ang II stimulation, APD(90) was prolonged by 22.1% (P < 0.01), myocyte volume, total protein content and membrane capacitance and CaN activity were significantly increased by 40.4%, 40.4%, 38.2% and 114.7% respectively (all P < 0.01) at 48 h after Ang II stimulation. HERG gene transfection upregulated I(HERG) tail current (3.6-fold higher than I(Kr)-rapidly activating delayed rectifier potassium current, P < 0.01). HERG gene transfection also accelerated and repolarization and a shortened APD(90) and inhibited myocyte hypertrophy and CaN activation induced by Ang II., Conclusions: Ang II induced prolongation of APD(90) is directly associated with myocyte hypertrophy by increasing the Ca(2+) influx and resulting in the increment of intracellular Ca(2+) and activation of CaN reaction pathway.

Objective: Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R., Methods: To study the function of the gene eag of Bacillus anthracis vaccine strain A16R, according to the sequence of Bacillus anthracis Ames strain, we designed primers and constructed a recombinant plasmid by the spectinomycin resistance cassette, upstream homologous fragment and downstream homologous fragment of eag cloned in tandem in pKSV7. We introduced the recombinant into A16R by electroporation and screened the mutant using the principle of homologous recombination. We checked the mutant using the PCR and proteomics., Results: We constructed the recombinant plasmid successfully and got the eag deletion mutant. PCR results showed the gene eag was deleted; SDS PAGE showed evident differences between prime strain and mutant strain. Two-dimensional gel electrophoresis results displayed three EA1 protein points of prime strain were absent in the mutant strain., Conclusion: We constructed eag deletion mutant of Bacillus anthracis vaccine strain A16R. This research will be helpful to study the functions of eag gene and the other important genes of Bacillus anthracis.

Objective: To identify the gene mutation in a Chinese family with congenital long QT syndrome (LQTS) and predict the changes of the secondary structure of the protein., Methods: Polymerase chain reaction and DNA sequencing were used to screen for KCNH2 mutation in the proband. After the mutation was identified, KCNH2 gene of the family members was screened by multiplex PCR with site-specific primers. Network analysis software was used to predict the secondary structure of the KCNH2 protein., Results: A novel heterozygous missense mutation of F463L(GenBank accession no.EU218526) located at the transmembrane domain S2 of KCNH2 was detected. The mutation did not result in the change of the transmembrane domain, but altered the hydrophobicity and secondary structure of the protein., Conclusion: The novel mutation identified in this study has enriched the GenBank data of ion channel gene mutation in LQTS. The changes of the secondary structure caused by the gene mutation were analyzed by Mfold and TMHMM software, which may help to understand LQTS.

We have investigated the methods and mechanisms for analysis of the channel kinetics parameters of voltage-gated potassium channels, HERG (Human ether-à-go-go related gene) channels, in the process of electrophysiological recording. The current of HERG K+ channels expressed in Xenopus oocytes was studied using a two-electrode voltage clamp technique, and the channel kinetics parameters were analyzed through compiling different pulse protocol and recording the current. Results showed: (1) The HERG K+ channels, under conditions of being activated with depolarized pulse, expressed an inward-rectified property attributing to rapid inactivation. The activation curve could be obtained through fitting the depolarized potential and the following peak amplitude of tail current, while the parameters of time-dependent activation was obtained through fitting different depolarized duration and the corresponding peak amplitude of tail current. (2) The I-V relationship still exhibit marked inward rectification. Tail current decay traces were fitted with a bi-exponential function to determine the time constants of the fast and slow components of current decay. (3) The inactivation of HERG channels is voltage-dependent. The inactivation process was isolated with two different three-pulse protocols, with which the inactivation curve and nearly linear I-V relationship were obtained, respectively. Thus, altough the kinetics properties of HERG channels were complicated, the channels kinetics could be indirectly analyzed through differently designed pulse protocols, which provided the basis for investigation on Alanine-scanning mutagenesis and agent action.

Aim: To explore a method of the stable and persistent expression of HERG(human ether-a-go-go-related gene) channels in Xenopus oocytes, and investigate the alteration of rest membrane potential of oocytes and electrophysiological properties of expressed channel in different culture duration., Methods: HERG mRNA for injection was prepared with in intro transcription using vector plasmid pSP64 containing HERG cDNA fragment. Expressed HERG current was recorded using standard two-microelectrode voltage-clamp technique., Results: (1) Functional channels, with electrophysiological properties consistent with those of HERG channels were persistently expressed in oocytes membrane with this method. Furthermore, channel current could be recorded stably in 10-15 days. (2) The negative value of rest membrane potential increased gradually in the 3, 6, and 9 days of culture, and then decreased in the 12 days. The potential of peak value of inward rectification shifted gradually to the positive direction in 3, 6 and 9 days, and recovered in 12 days. Half-maximal activation potential (V1/2) of heterological expressed current shifted gradually to the negative direction in 3, 6 and 9 days of culture and then recovered in 12 days, the tendency of change was coincident with that of membrane rest potential., Conclusion: The investigation provides a method of persistent expression of HERG channel in Xenopus oocytes and offers evidences for the difference of electrophysiological experimental data of studies of molecular site and drugs effect of HERG channel in different experimental conditions.

Rapidly activating component of delayed rectifier potassium current (I(Kr)) plays a key role in the repolarization phase of cardiac action potential. Human ether-a-go-go-related gene (HERG) encodes the alpha subunit of this potassium channel. Mutations of HERG gene induce genetic long QT syndrome (LQTS). Furthermore, I(Kr)/HERG is the target of some drugs which may cause cardiac QT interval prolongation. Some other drugs with different chemical structures also may block the channel and prolong QT interval, which even developed into acquired arrhythmias. This review summarized the recent progress of structure, gating mechanisms and functions of I(Kr)/HERG channel, I(Kr)/HERG related arrhythmias, interaction between K+ channel and drugs, and strategies of grading-up the I(Kr)/HERG target.

Objective: To investigate the functional expression of HERG mutation A561V detected in a Chinese congenital long QT syndrome family., Methods: The mutation gene A561V was cloned into eukaryotic expressive vector pcDNA3 by quick site-directed mutagenesis PCR and restriction enzymes. The wild-type HERG, heterozygous type HERG and HERG mutation A561V were respectively cotransfected with pRK5-GFP into HEK293 cells by Suprefact transfection regent. The protein expression was measured by immunofluorescence method and Western blot. The electrophysiological characteristics of transfected cells were determined by whole cell patch-clamp technique., Results: Direct sequence analyses revealed a C to T transition at position 1682. A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein expression of mutation and heterozygosis were located in cytoplasm and cellular membrane. 155 kDa and 135 kDa protein bands were detected in wild type HERG channel while only 135 kDa protein band was shown in heterozygous and mutational channels. Significant HERG tail-current was recorded in wild type HERG channel but not in mutation and heterozygosis channels., Conclusion: This study evidenced a functional dominant-negative current suppression in HEK293 cells transfected with HERG mutation A561V.

Because HERG potassium channel has important effects on both proarrhythmia and antiarrhythmia, we use immunofluorescence and Western blotting methods to detect the expression of HERG channel of HERG-HEK cells in different concentrations of matrine, oxymatrine and resveratrol. The findings showed that both matrine (1 micromol x L(-1) ) and oxymatrine ( 1micromol x L (-1) ) increased HERG channel expression ( n = 5, P < 0. 05 ) , while matrine (100 micromol x L(-1) ) decreased HERG channel expression ( n = 5, P < 0. 05), resveratrol didn't affect HERG channel expression. In conclusion, different concentrations of matrine and oxymatrine affect HERG channel expression, while there is no relationship between resveratrol and HERG channel expression. It provides a theoretical support for the safety and mechanism of anti-arrhythmic drugs.

Objective: To investigate the protocol of the construction of HERG gene mutations, an A561V mutation which was detected in a Chinese congenital long QT syndrome (LQTS) family had been constructed and expressed in vitro., Methods: The A561V cloning vector PGEM-HERG-A561V was constructed by quick site-directed mutagenesis PCR. The A561V expressive vector pcDNA3-HERG-A561V was constructed by restriction enzymes. pRK5-GFP was cotransfected with pcDNA3-HERG-A561V or wild type pcDNA3-HERG into HEK293 cells by Superfect transfection reagent. The protein was measured by immunofluorescence., Results: Direct sequence analyses revealed a C to T transition at position 1682. The A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein of mutation was expressed in cytoplasm and cellular membrane while the wild type gene was expressed only on cellular membrane., Conclusion: The protocol can be used successfully to construct and express HERG A561V mutation and it forms the basement of the further study on functions of mutation.

Objective: Hereditary long QT syndrome (LQTS) is a cardiac disorder characterized by prolongation of QT interval on electrocardiograms (ECGs) and syncope and sudden death caused by a specific multi-polymorphic ventricular tachyarrhythmia known as torsade de pointes. LQTS is caused by mutations in cardiac sodium channel gene SCN5A; potassium channel subunit genes KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2; calcium channel gene Cav2.1. and ankyrin-B gene ANK2., Methods: We characterized 77 Chinese LQTS patients with clinical manifestations and mutations in the main LQTS genes, KCNQ1 and KCNH2 using PCR and sequence analysis., Results: The spectrum of ST-T-wave patterns of 24 (31.2%) probands were considered as LQT1, 42 (54.5%) as LQT2 and 3 (3.9%) as LQT3. The remaining 8 (10.3%) could not be characterized. The average age for this population of LQTS patients was (27.6 +/- 16.4) years and the average QTc (561 +/- 70) ms, and the age of the first syncopal attack was (17.6 +/- 14.7) years. The triggering factors for cardiac events happening in these mutation carriers included physical exercise, emotional excitement and auditory irritation. We identified 4 KCNQ1 mutations and 7 KCNH2 mutations. Six of them were first identified with some data already shown. In this paper we showed the data of 6 other mutations., Conclusions: LQT2 is the most common type of LQTS in Chinese; 2 mutations of KCNQ1 and KCNH2 were first identified in this report; there are some differences between Chinese and North American or European LQTS patients in clinical characters and ECG.

Objective: To identify the electrophysiological properties of long-QT syndrome (LQTS) associated missense mutations in the outer mouth of the HERG potassium channel in vitro., Methods: Mutations V630A and N633S were constructed by Megaprimer PCR method and cRNA were produced by T7 RNA polymerase. The electrophysiological properties of the mutation were investigated in the Xenopus oocyte heterologous expression system., Results: Coexpression of mutant and wild-type HERG subunits caused a dominant-negative effect, and the currents were significantly decreased. Compared with wild-type HERG channels, V630A and N633S mutations were related to decreased time constants for inactivation for V630A/WT and N633S/WT at all potentials, reduced slope conductance and the voltage dependence of steady-state inactivation was shifted to negative potentials for V630A/WT and N633S/WT., Conclusion: Present study shows that LQTS associated missense mutations located in the outer mouth of HERG cause a dominant-negative effect and alterations in steady-state voltage dependence of channel gating of heteromultimeric channels suggesting a reduction in expressional current might be one of the pathophysiologic mechanisms of LQTS.

Background & Objective: Previous studies have found that HERG, a kind of K(+) channel protein, is expressed in some cancers, but its expression is weak or lost in normal tissues. This study was to investigate HERG expression in various cancer cell lines, its correlation with chemosensitivity of cancer cells to doxorubicin, and its biochemical modulation., Methods: HERG expression in human colon carcinoma cell line HT-29, human breast carcinoma cell line MCF-7, human lung carcinoma cell line A549, and human high-metastatic giant cell lung carcinoma cell line PG was analyzed by Western blot. After transfection of herg gene, the cytotoxicity of doxorubicin, erythromycin (a HERG K+ channel blocker), or doxorubicin in combination with erythromycin to cancer cells was analyzed by MTT assay. Intracellular content of doxorubicin was observed under fluorescent microscope., Results: HERG protein level was higher in HT-29, MCF-7, and PG cells than in A549 cells. A549 cells were more sensitive to doxorubicin than HT-29 cells. The 50% inhibitory concentration (IC(50)) of doxorubicin was obviously higher in herg-transfected A549 cells than in parent A549 cells. Erythromycin obviously suppressed the growth of HT-29 cells, and showed synergic cytotoxicity with doxorubicin to HT-29 cells. There is no difference in intracellular doxorubicin content among herg-transfected A549 cells, pCDNA3.1-transfected A549 cells, and parent A549 cells., Conclusions: HERG expression might negatively correlate with the chemosensitivity of tumor cells to doxorubicin. Erythromycin may act as a modulator, and has synergic effect with doxorubicin. HERG may serve as a molecular marker and modulating target for individualized cancer therapy.

Objective: To investigate the molecular pathology in families with long QT syndrome (LQTS) including Jervell-Longe-Nielsen syndrome (JLNS) and Romano-ward syndrome (RWS) and Brugada syndrome (BS) in Chinese population., Methods: Polymerase chain reaction and DNA sequencing were used to screen for KCNQ1, KCNH2, KCNE1, and SCN5A mutation., Results: We identified a novel mutation N1774S in the SCN5A gene of the BS family, a novel mutation G314S in a RWS family which had also been found in Europe, North America, and Japan, and a single nucleotide polymorphisms (SNPs) G643S in the KCNQ1 of the JLNS family. In this JLNS family, another heterozygous novel mutation in exon 2a was found in KCNQ1 of the patients., Conclusion: New mutations were found in our experiment, which expand the spectrum of KCNQ1 and SCN5A mutations that cause LQTS and BS.