Aminoglycosides Saved A Nonsense Mutation In The Herg Channel Drug

BackgroudLong QT syndrome is an inherited arrhythmia syndrome characterized by delayed cardiac repolarization and prolonged QT interval,abnormal T wave and U wave on the ECG,and recurrent ventricular arrhythmia and torsades de pointes which can lead to syncope,sudden cardiac death and a series of cardiac events.Until now,twelve genes have been identified to be associated with long QT syndrome,among these genes eight encode the ion channels,others encode proteins interacting with ion channels.The human ether-a-go-go-related gene(HERG) encodes the a-subunit of the rapidly activating component of the delayed rectifier potassium channel current(I_Kr). Mutations in HERG have been proven to result in type 2 long QT syndrome(LQT2) by a "loss-of-function" mechanism,and LQT2 accounts for about 45%in all types of long QT syndrome.In over 300 HERG mutations linked to LQT2,62%are missense mutations,30%are nonsense or frameshift mutations and 8%are other mutations. Previous studies have demonstrated that most LQT2 linked missense mutations reduce functional HERG current by a "trafficking deficient" mechanism.This trafficking defect could be rescued by either incubation at lower temperatures(27℃) or treatment with drugs such as HERG channel blockers(E4031,cisapride) and other compounds(thapsigargin).Unlike missense mutations,nonsense mutations always introduce premature termination codons,giving rise to toxic truncated proteins that exert a dominant negative effect on wild type channel.Currently,there are no reports regarding whether these truncated HERG proteins can be pharmacologically rescued to express full-length functioning proteins.Aminoglycoside antibiotics are a group of promising drugs which are known to have bacteriocidal activity and even are able to inhibit the premature termination codons,reduce the accuracy and fidelity of translation,and encourage the expression of functional proteins-a phenomenon observed in cystic fibrosis,Duchenne muscular dystrophy,diabetes insipidus and other inherited diseases resulting from nonsense mutations.Extensive studies in heterologous expression systems,animal models,and clinical trials have demonstrated that aminoglycoside antibiotics can suppress the nonsense mutation and partially restore functional full-length expression by permitting the readthrough of premature termination codons.In 1985,G-418 was shown to suppress a nonsense mutation and restore gene activity to almost 25%of WT levels.The effect of gentamicin on readthrough efficacy has also been well documented.ObjectivesThe study was designed to:(1) examine the expression of nonsense mutant HERG channels;(2) investigate the effect of aminoglycoside antibiotics G-418 and gentamicin on homozygous HERG mutations leading to LQT2 in a heterologous expression system,and analyze the reason why different mutants responded variably to the treatment of a certain aminoglycoside antibiotics;(3) evaluate the pathogenesis mechanism of HERG nonsense mutations resulting in LQT2;(4) explore the effect of aminoglycoside antibiotics G-418 and gentamicin on heterozygous HERG mutations; (5) investigate the effect of aminoglycoside antibiotics on the trafficking defect of missense mutation.MethodsFour HERG C-terminal nonsense mutants(R1014X,W927X,R863X and E698X) were constructed by polymerase chain reaction based mutagenesis strategy.All mutations were verified by sequencing and then subcloned into pcDNA3.1 vector. The wild type HERG and four mutant HERG plamids were amplified and purified, then transiently transfected into HEK293 cells.Green fluorescent protein gene was co-transfected as an indicator.Prepare the bath solution and the internal pipette solution for HERG channel current recording,set the parameters for stimulating the HERG current-voltage curve.Channel currents were recorded by whole-cell patch clamp after transfection for 36～48 hours.Pharmacological rescue was applied by culturing the cells in 400μg/mL G-418 or gentamicin for 24 hours.Cells were cultured in drug-free medium for 1 hour before patch-clamp recordings.Western blot was performed to test the expression of wild type and mutant HERG channel proteins.Anti-HERG N-terminal antibody was used before drug treatment, C-terminal antibody was used to evaluate the effect of drugs on the mutant channel proteins.Immunofluorescence was applied to detect the distribution pattern of wild type and mutant HERG channel proteins under a confocal laser scanning microscope. Anti-HERG N-terminal antibody was used before drug treatment,C-terminal antibody was used to evaluate the effect of drugs on the intracellular distribution of mutant channel proteins. Results1 The establishment of HEK 293 cell expression system:HEK 293 cell adhered and grew well after continuous passage,looking like epithelioid cells.The day before transfection,about 1×10⁵ cells were cultured in 35mm dishes.It was time for transfection when the confluence rate was 50%～70%.The transient transfection efficiency was ranging from 50%to 60%after transfection for 36～48 hours.The positively transfected HEK 293 cells displayed green fluorescence,while the untransfected cells showed no fluorescence.Patch clamp recordings were performed on the cells with green fluorescence,distinct edge,smooth surface,moderate size.2 The current expression of HERG nonsense mutations:Wild type HERG channel demonstrated the typical characteristics of voltage depedent activation and inward rectification,with peak tail current density of 47.8±6.3 pA/pF(n=12).Both R1014X and W927X mutants displayed HERG like currents,however,the mean peak tail current densities of R1014X(3.9±1.4 pA/pF,n=8,P<0.01) and W927X(11.6±2.4 pA/pF,n=8,P<0.01) channels were significantly lower than that of wild type channel. R863X and E698X mutants generated currents comparable to endogenous currents in untransfected HEK 293 cells.No tail currents were observed in at least eight cells, illustrating their inability to form functional channels.3 The effect of aminoglycoside antibiotics on homozygous HERG mutations: Aminoglycoside antibiotics increased the functional expression of R1014X and W927X mutants.After treated by 400μg/ml G-418 and gentamicin,the peak tail current density increased to 12.7±3.3 pA/pF(n=7,P<0.05) and 18.3±3.7 pA/pF(n=8, P<0.05).In addition,the leftward shift of the R1014X channel activation curve was corrected by drug treatment,indicating that electrophysiological properties of the mutant channel were partially restored.Similar results were observed on the W927X mutant,its density was enhanced to 19.5±2.7 pA/pF(n=7,P<0.05).The voltage dependence of activation did not differ among WT,W927X and gentamicin-treated W927X mutant channel.However,gentamicin had no effect on the upstream mutations,R863X and E698X,as no tail current could be observed after drug treatment.4 The expression and the intracellular distribution of nonsense mutant HERG channel proteins:The expression of WT and mutant HERG channel proteins was analyzed by western blotting using an anti-HERG N-terminal antibody.Two bands at around 135 kDa and 155 kDa were observed in the WT-HERG lane,representing the core-glycosylated immature form and the complex-glycosylated mature form of HERG channel proteins,respectively.Both R1014X and R863X expressed truncated HERG proteins.R1014X showed two bands at around 120 kDa and 140 kDa, signifying the presence of core and complex glycosylation.However,R863X only showed a single 90 kDa band,indicating that the trafficking defect may have inhibited the acquisition of complex glycosylation.To test subcellular localization of wild type and mutant HERG channels,confocal imaging was performed using an N-terminal antibody.No fluorescence was detected in untransfected HEK293 cells.In cells expressing wild type HERG,strong red fluorescence signals were visualized predominantly in the plasma membrane.Cells transfected with R1014X and W927X mutants demonstrated a fluorescence distribution pattern similar to wild type HERG cells.In contrast,cells transfected with R863X and E698X mutants displayed a restricted perinuclear distribution.5 The effect of aminoglycoside antibiotics on nonsense mutant HERG channel proteins:An anti-HERG C-terminal antibody,which is incapable of recognizing truncated channel proteins,was used to test the effect of the drugs.Two bands at around 135 kDa and 155 kDa were observed in cells expressing wild type HERG, whereas no protein bands were observed in cells transfected with R1014X mutant. After treatment with G-418 or gentamicin,however,two protein bands were detected, implying translational readthrough for full-length HERG channel proteins.In contrast, the trafficking defect of N470D apparently could not be rescued,as only one protein band was visible following drug treatment.The C-terminal antibody was used to test the effect of the drugs on the distribution pattern of mutant channel proteins.No fluorescence was detected in untransfected HEK 293 cells.In HEK 293 cells expressing the R1014X mutant,gentamicin induced red fluorescence signals,indicating the appearance of full-length HERG channel proteins.However,no fluorescence signals were observed before and after gentamicin treatment in R863X mutant.These observations were in accordant with the results in western blot analysis.6 The effect of aminoglycoside antibiotics on dominant negative effect:Since LQT2 is inherited in an autosomal dominant pattern,patients generally have a heterozygous genotype.Therefore,further study was performed to test on heteromultimeric channels by co-transfection of WT and mutant HERG genes.The tail current density of WT/R1014X channels was less than half of that from WT channel alone(13.1±2.2 pA/pF,n=8 vs 47.8±6.3 pA/pF,n=12,P<0.05),indicative of "dominant negative" inhibition.In the cells co-transfected with WT/R1014X,gentamicin and G-418 demonstrated different results:gentamicin enhanced the expression of WT/R1014X channels by increasing the average tail current density 2.2-fold(29.2±3.7 pA/pF,n=12,P<0.05). However,current density remained at the same level following G-418 treatment (13.7±1.8pA/pF,n=8,P>0.05).It is noteworthy that G-418 and gentamicin treatment did not shift the activation curve of WT/ R1014X channels.Conclusions1 The functional expression of C-terminal HERG nonsense mutation is dependent on the length of amino acids of the C-terminal truncation.2 Aminoglycoside antibiotics enhanced the functional expression of homozygous mutant HERG channels,restored its electrophysiological characteristics.3 Aminoglycoside antibiotics inhibited the premature stop codons,allowed the readthrough of the translation and partially restored the expression of functional full-length HERG channel proteins.4 Aminoglycoside antibiotics diminished the dominant negative effect of nonsense mutant channel,enhanced the he functional expression of heterozygous mutant HERG channels.