Progress in research on defective protein trafficking and functional restoration in HERG-associated long QT syndrome / 中华医学遗传学杂志

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.

Expression of human ether-a-go-go-related gene in laryngeal carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the expression of human ether-α-go-go-related gene (herg) and hERG protein expressed by the gene in laryngeal carcinoma compared with the control group(mucosa adjacent to cancer of 2 cm).@*METHOD@#Expression of herg and hERG protein was detected by immunohistochemistry (SP) and real-time PCR in resected tissue of laryngeal carcinoma and mucosa adjacent to cancer of 2 cm.@*RESULT@#(1) By immunohistochemistry, the positive expression rate of hERG in laryngeal carcinoma was 76.7% (23/30), while it was 10.0% (2/20) in mucosa adjacent to cancer of 2 cm, the difference between which was statistically significant (P < 0.05). (2) By real-time PCR, the expression level of herg mRNA in laryngeal carcinoma is 2.25 times higher than that in mucosa adjacent to cancer of 2 cm.@*CONCLUSION@#Herg is highly expressed in tissue of laryngeal carcinoma, and it may be have some relevance to the happening and development of laryngeal carcinoma.

Clinical characteristics of patients with congenital long QT syndrome and bigenic mutations / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Congenital long QT syndrome (LQTS) is an ion channelopathy associated with genetic mutations. It is well known that most LQTS patients (91%) have a single mutation. The purpose of this study was to investigate the clinical characteristics of congenital LQTS patients with bigenic mutations in Taiwan, China.</p><p><b>METHODS</b>Congenital LQTS patients were recruited consecutively at Taiwan University Hospital in Taiwan from 2003 to 2009. The diagnosis of LQTS was defined by an LQTS Schwartz score greater than 4. Mutation screening in KCNQ1, KCNH2, KCNE1, and SCN5A was performed using direct sequencing.</p><p><b>RESULTS</b>Three of 16 LQTS patients (18.7%) were identified with bigenic mutations. One patient had missense mutations in KCNQ1 and KCNH2, the second in KCNQ1 and KCNE1, and the third in KCNH2 and SCN5A. The mean age at onset of LQTS for patients with bigenic mutations was (17 ± 3) years, and all of these patients were female. Two of them experienced seizure and one presented with syncope, although one of them had a family history of syncope. The mean QTc interval was (515 ± 17) ms, similar to those with single mutation or SNPs ((536 ± 74) ms, P = 0.63). Compared to those LQTS patients with single mutation or SNPs, a significantly higher percentage of LQTS patients with bigenic mutations presented with seizure and were younger at onset of the first index event (P = 0.03 and 0.001, respectively), but lower percentage of them presented with sudden cardiac death (P = 0.03).</p><p><b>CONCLUSIONS</b>Although the percentage of bigenic mutations in LQTS is less than 10% in Caucasian populations, we identified 3 of 16 LQTS patients (18.7%, 95% confidence interval: 0.04-0.46) with bigenic mutations in Taiwan. However, the severity of their clinical presentations was not higher than those patients with single mutation or SNPs.</p>

A novel deletion-frameshift mutation in the S1 region of HERG gene in a Chinese family with long QT syndrome / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The congenital Long QT syndrome (LQTS) is a hereditary cardiac channelopathy that is characterized by a prolonged QT interval, syncope, ventricular arrhythmias, and sudden death. The chromosome 7-linked type 2 congenital LQTS (LQT2) is caused by gene mutations in the human ether-a-go-go-related gene (HERG).</p><p><b>METHODS</b>A Chinese family diagnosed with LQTS were screened for KCNQ1, HERG and SCN5A, using polymerase chain reaction (PCR), direct sequencing, and clong sequencing. We also investigated the mRNA expression of the HERG gene.</p><p><b>RESULTS</b>We identified a novel I414fs + 98X mutation in the HERG gene. The deletion mutation of 14-bp in the first transmembrane segment (S1) introduced premature termination codons (PTCs) at the end of exon 6. This mutation would result in a serious phenotype if the truncated proteins co-assembled with normal subunit to form the defective channels. But only the proband was symptomatic.</p><p><b>CONCLUSIONS</b>We found that the mRNA level of the HERG gene was significantly lower in I414fs + 98X carriers than in noncarriers. We found a novel I414fs + 98X mutation. The mRNA level supports that NMD mechanism might regulate the novel mutation.</p>

Effect of berberine, liensinine and neferine on HERG channel expression / 中国中药杂志

<p><b>OBJECTIVE</b>Immunofluorescence and Western blot methods were adopted for qualitative and quantitative detections of the effect of different concentrations of berberine, liensinine and neferine on the expression of stable transfection in HERG potassium channel in HEK-293 cells, as well as the effect of different concentrations of berberine on protein expression of Ikr channel in cardiac muscular tissues, in order to investigate the anti-arrhythmic mechanism of berberine, liensinine and neferine.</p><p><b>METHOD</b>Western blot method was used to detect protein expression of HERG channel in HERG-HEK cells. Immunofluorescence method as well as confocal laser microscope were used to detect the effect of different concentrations of berberine, liensinine and neferine on protein expression of HERG channel. Western blot method was used to detect the effect of different concentrations of berberine on protein expression of Ikr channel in cardiac muscular tissues as well as the effect of berberine, liensinine and neferine on protein expression of stable transfection in HERG potassium channel in HEK-293 cells.</p><p><b>RESULT</b>Western blot experiment manifested that stable transfection of HEK293 cells containing HERG genes could increase protein expression of HERG channel. Berberine (10, 30 micromol x L(-1)) remarkably inhibited protein expression of HERG channel in HERG-HEK cells (P < 0.01). Berberine (10, 20 mg x kg(-1)) also inhibited protein expression of Ikr channel in rat ventricular tissues (P < 0.05). Liensinine (3, 10, 30 micromol x L(-1)) increased protein expression of HERG channel in HERG-HEK cells (P < 0.05). Neferine showed no effect on protein expression of HERG channel in HERG-HEK cells.</p><p><b>CONCLUSION</b>The stably transfection of HERG-HEK cells can increase protein expression of HERG channel. Berberine shows inhibitory effect on protein expressions of in vitro HERG-HEK cells and Ikr channel in rat ventricular tissues. Liensinine improves protein expression of HERG channe in HERG-HEK cells. Neferine shows no effect on protein expression of HERG channel.</p>

KCNQ1, KCNH2, KCNE1 and KCNE2 potassium channels gene variants in sudden manhood death syndrome / 法医学杂志

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.

Frequency- and state-dependent blockade of human ether-a-go-go-related gene K+ channel by arecoline hydrobromide / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The rapidly activating delayed rectifier potassium current (I(Kr)), whose pore-forming alpha subunit is encoded by the human ether-a-go-go-related gene (hERG), is a key contributor to the third phase of action potential repolarization. The aim of this study was to investigate the effect and mechanism of arecoline hydrobromide induced inhibition of hERG K(+) current (I(hERG)).</p><p><b>METHODS</b>Transient transfection of hERG channel cDNA plasmid pcDNA3.1 into the cultured HEK293 cells was performed using Lipofectamine. A standard whole-cell patch-clamp technique was used to record the I(hERG) before and after the exposure to arecoline.</p><p><b>RESULTS</b>Arecoline decreased the amplitude and the density of the I(hERG) in a concentration-dependent manner (IC(50) = 9.55 mmol/L). At test potential of +60 mV, the magnitude of I(hERG) tail at test pulse of -40 mV was reduced from (151.7 ± 6.2) pA/pF to (84.4 ± 7.6) pA/pF (P < 0.01, n = 20) and the magnitude of I(hERG) tail at test pulse of -110 mV was reduced from (-187.5 ± 9.8) pA/pF to (-97.6 ± 12.6) pA/pF (P < 0.01, n = 20). The blockade of arecoline in the open and inactivated state was significant in a state-dependent manner. The maximal blockade was achieved in the inactivated state. Studies of gating mechanism showed that the steady-state activation curve of I(hERG) was significantly negatively shifted by arecoline. Time constants of activation were shortened. Steady-state inactivation curve and time constants of fast inactivation were not significantly affected by arecoline. Furthermore, the inhibition of I(hERG) by arecoline was characterized markedly by a frequency-dependent manner from 0.03 to 1.00 Hz pulse.</p><p><b>CONCLUSION</b>Arecoline could potently block I(hERG) in both frequency and state-dependent manner.</p>

Lidamycin inhibits the proliferation of HERG K+ channel highly expressing cancer cells and shows synergy with anticancer drugs / 药学学报

This study is to investigate inhibitory effects of lidamycin (LDM) on the proliferation of HERG K+ channel highly expressing cancer cells and its synergy with anticancer drugs. MTT assay was used to examine the inhibitory effects of lidamycin combined with various anticancer drugs on the proliferation of human lung cancer A549 cells, human colon cancer HT-29 cells and herg-stably-transfected A549 cells. Using the xenograft model of subcutaneously transplanted HT-29 in nude mice, inhibitory effect was appraised in vivo. The coefficient of drug interaction (CDI) was used to evaluate the synergistic effect of drug combination. LDM significantly inhibited the proliferation ofA549 cells and HT-29 cells with IC50 values of 2.14 and 4.64 ng mL(-1), respectively. The efficacy in HT-29 cells with high HERG potassium expression level is less potent than that in A549 cells with low expression level. In terms of IC50 values, LDM suppressed the growth of herg-stably-transfected A549 cells less potently than pCDNA3.1-stably-transfected A549 cells. There existed synergistic effects in the combinations of fluorouracil (5-FU) and LDM, doxorubicin (DOX) and LDM, or hydroxycamptothecine (HCPT) and LDM. CDI values of the combinations of 5-FU and LDM were more than 0.75. CDI values of LDM and DOX were more than 0.70, but some CDI values of LDM and HCPT were less than 0.70. As for the CDI values, synergistic effects of the combination of LDM and HCPT were the most potent of the three groups. There is no relationship between the inhibitory effect of the growth of cancer cells by 5-FU and HERG potassium expression level. HERG expression level negatively correlated with inhibitory effect on the proliferation of cancer cells by DOX. HERG expression levels and chemosensitivity were positively correlated for HCPT. In the model of subcutaneously xenograft transplanted HT-29 in vivo, LDM and/or HCPT effectively inhibited the growth of HT-29 in nude mice, and the optimum CDI of the combination of LDM and HCPT was less than 1. HERG expression level negatively correlates the chemosensitivity of cancer cells to LDM. There exist synergistic effects in vitro and in vivo in the combination of LDM and HCPT, which inhibitory effects of the proliferation of cancer cells positively modulated by HERG potassium expression level. HERG K+ channel may become a target of combined therapy for choosing anticancer drugs.

A novel mutation of the KCNH2 gene in a family with congenital long QT syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To perform mutation analysis in a family with long QT syndrome.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effect of matrine on human ether à go-go related gene (HERG) channels expressed in Chinese hamster ovary cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effect of matrine on human ether à go-go related gene (HERG) potassium channels expressed in Chinese hamster ovary (CHO) cells and investigate whether HERG channel is a new target of the pharmacological effect of matrine on arrhythmia and tumor</p><p><b>METHODS</b>HERG channel potassium current in CHO cell was recorded using whole-cell patch-clamp technique, and the influence of matrine on the current was explored.</p><p><b>RESULTS</b>Matrine inhibited HERG potassium current in a dose-dependent manner, and the 50% inhibitory concentration (IC IC(50)) was 411±23 μmol/L. Matrine had no significant effect on the activation kinetics, and mainly blocked HERG channels in their closed state.</p><p><b>CONCLUSIONS</b>The blocking effect of matrine on HERG channels might be one of the mechanisms against arrythmias and tumors. Unlike most other blockers exerting blocking effect at the intracellular sites by entering the cell with the opening of HERG channel, matrine blocked HERG channels at the extracellular sites.</p>

Gene mutation analysis of a Chinese family of congenital long Q-T syndrome type three / 中华儿科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Eukaryotic expression vector pcDNA3-HERG transfection inhibits angiotensin II induced neonatal rabbit ventricular myocyte hypertrophy in vitro / 中华心血管病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Comparative pharmacophore analysis of dual dopamine D2/5-HT(2A) receptor antagonists / 药学学报

Dual dopamine D2/5-HT2A receptor antagonists have potent activity and are referred to atypical antipsychotics due to their lower propensity to elicit EPS and their moderate efficacy toward negative symptoms. However, an on-going challenge in developing atypical antipsychotics drugs is to maintain the favorable profiles and avoid of cardiovascular risk. In this paper, comparative pharmacophore analysis of dual dopamine D2/5-HT2A receptor antagonists, hERG K+ channel blockers, and alA adrenoceptor antagonists is carried out, and the results could give some insight into multi-target drug design.

Identification of a novel KCNH2 mutation in a family with congenital long QT syndrome and prediction of the secondary structure of its encoding protein / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Analysis and analyzing mechanisms of HERG channel kinetics / 生物医学工程学杂志

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.

Mutation screening in KCNQ1, HERG, KCNE1, KCNE2 and SCN5A genes in a long QT syndrome family

<p><b>INTRODUCTION</b>Long QT syndrome (LQTS), an inherited cardiac arrhythmia, is a disorder of ventricular repolarisation characterised by electrocardiographic abnormalities and the onset of torsades de pointes leading to syncope and sudden death. Genetic polymorphisms in 5 well-characterised cardiac ion channel genes have been identified to be responsible for the disorder. The aim of this study is to identify disease-causing mutations in these candidate genes in a LQTS family.</p><p><b>MATERIALS AND METHODS</b>The present study systematically screens the coding region of the LQTS-associated genes (KCNQ1, HERG, KCNE1, KCNE2 and SCN5A) for mutations using DNA sequencing analysis.</p><p><b>RESULTS</b>The mutational analysis revealed 7 synonymous and 2 non-synonymous polymorphisms in the 5 ion channel genes screened.</p><p><b>CONCLUSION</b>We did not identify any clear identifiable genetic marker causative of LQTS, suggesting the existence of LQTS-associated genes awaiting discovery.</p>

Functional expression of congenital long QT syndrome related HERG mutation A561V in vitro / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the functional expression of HERG mutation A561V detected in a Chinese congenital long QT syndrome family.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>This study evidenced a functional dominant-negative current suppression in HEK293 cells transfected with HERG mutation A561V.</p>

Effects of matrine, oxymatrine and resveratrol on HERG channel expression / 药学学报

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.

Construction of eukaryotic expression vector of congenital long QT syndrome related HERG gene A561V mutation and its expression in HEK293 cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Electrophysiological characterization of long QT syndrome associated mutations V630A and N633S / 中华心血管病杂志

<p><b>OBJECTIVE</b>To identify the electrophysiological properties of long-QT syndrome (LQTS) associated missense mutations in the outer mouth of the HERG potassium channel in vitro.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>