Propofol Inhibits Currents Of Both Human Ether-a-go-go-related Gene And Its Non-sense Mutation, Q738X, In HEK293 Cells

There are two types of long QT syndrome (LQTS), inherited and acquired LQTS. Inherited LQTS is caused by mutation of genes encoding cardiac ionic channels or associated partners, which leads to dysfunction of corresponding ionic channel proteins. So far, molecular genetic studies have discovered at least ten forms of inherited LQTS numbered in order of discovery as LQT1-LQT10. Mutation in the hERG gene accounting for LQT2 is one of the principal causes of inherited LQTS. The human ether-a-go-go-related gene (hERG) encodes the a-subunit of the rapidly activating delayed rectifier potassium ion channel underlying IKr-and this, current is essential to the repolarization phase of cardiac action potential in the mammalian heart. More recently, Yasuda et al. reported a small family of hereditary LQT2 caused by a novel hERG mutation, showing a wide variety of ECG phenotypes among family members under the common single nucleotide mutation (c.C2212T and p.Q738X). However, the mechanism for hERG channel dysfunction in the Q738X mutation has not been studied.Acquired LQTS is mainly induced by drugs. The drug-induced LQTS can result from:1. direct block of channel conduction. The drug has high affinity with the particular aromatic amino acid (position Y652 and F656) located on the S6 transmembrane domain of the hERG channel. When it binds with the hERG channel, it leads to K+ efflux decrease in phase 3 repolarization of cardiac cells, and therefore prolongs the length of QT interval.2. Indirect inhibition by disrupting channel protein trafficking. The drug disrupts normal hERG channel protein processing and maturation to reduce its surface membrane expression.Propofol is a short-acting intravenous anesthetic agent widely used for the induction and maintenance of general anesthesia and for sedation in intensive care units. Despite its commendable record, there have been scattered reports of an association of propofol use with sudden death. Several recent studies investigated the effect of poprofol on QT interval, however these results were conflicting. Some studies reported that propofol prolonged the QT interval. Conversely, other studies found that propofol had no effect or shortened the QT interval. In these studies propofol was administered simultaneously with other agents within a short period of time; therefore, it is difficult to determine its selective effect on the QT interval. To our knowledge, the effect and mechanism of poprofol on the QT interval are not entirely clear but could involve actions on the hERG K+ channel.So, the objectives of this study are as follows:1. To examine the electrophysiological consequences of the Q738X mutation; 2. To evaluate the effect of propofol on reconstituted wild type (WT) and its underlying mechanism; 3. To evaluate the effect of propofol on mutant hERG channels using the heterologous expression system in HEK-293 cells.Mutant Q738X, Y652A and F656C-hERG were constructed by overlap extension PCR and were verified by DNA sequencing, respectively.HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified 5% CO2 incubator at 37℃.For transfection of hERG constructs in different amounts, cells were plated in a culture dish and transfected transiently 24～36 h later with the calcium phosphate precipitation method or lipofectamine method. Transfected cells were transferred to a bath mounted on the stage of an inverted microscope. First, we studied the current, current-voltage relation, actvation, deactivation and inactivation of WT-hERG and WT/Q738X-hERG channel. Secondly, we observed the effect of propofol on WT-hERG, Y652A, F656C, and WT/Q738X-hERG channel, respectively.The cells were scrapped in ice-cold phosphate-buffered saline and lysed in RIPA buffer. Protein concentrations were determined by the bicinchoninic acid method. Protein per sample was electrophoresed on 10% Tris-acetate gels and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk before incubation with rabbit anti-hERG antibody (1:200) overnight. The membranes were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10000) in TBST for 1 h at room temperature. After washing with TBST, the membranes were developed using ECL. Blots were analyzed and quantified by Quantity one software.At 36～48 h after transfection, HEK293 cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA) at room temperature. Cells were then labeled with rabbit polyclonal anti-hERG and chicken polyclonal anti-calreticulin at 4℃overnight followed by incubation with FITC-conjugated goat antirabbit IgG secondary antibody and Alexa fluor-conjugated goat antichicken IgG secondary antibody at 37℃for 2 h.Immunofluorescence staining was viewed with a confocal laser scanning microscope (excitation and emission wavelength for FITC were 488 and 520nm, respectively; excitation and emission wavelength for Alexa fluor were 633 and 647 nm, respectively).Data are presented as mean±S.E.M. The data were acquired with use of Pulse 8.67 software and analyzed by SPSS 13.0 and Origin 6.0 software. Statistical comparisons were evaluated by t tests and one-way ANOVA. P values of less than 0.05 were taken as significant. There were classic currents from HEK-293 cells transfected with WT-hERG, but no currents from cells transfected with Q738X-hERG channel. These cells cotransfected with WT/Q738X-hERG showed currents with similar waveforms but reduced current amplitudes compared to those cells expressing WT-hERG channels alone. The peak tail current of WT-hERG (4μg), WT-hERG (2μg) and WT/Q738X (2μg each) was 59.9±3.2 pA/pF,32.2±1.7 pA/pF and 26.2±3.5 pA/pF (P<0.05 vs 4μg WT-hERG), respectively. Therefore, we conclude the Q738X mutation had no a potent dominant-negative effect on WT-hERG channel properties.Mutant Q738X had not altered the kinetic features of WT-hERG channel (activation, deactivation and inactivation).The results of Western blot showed two bands (135 and 155 KDa) in WT-hERG group, only one 135 KDa band in Q738X-hERG group, and both bands, but a weaker 155 KDa band in WT/Q738X-hERG group.The results of confocal imaging showed WT-hERG mainly expressed in membrane, Q738X-hERG expressed in cytoplasm and WT/Q738X-hERG expressed in both membrane and cytoplasm.Propofol inhibited WT-hERG channel current in a concentration-dependent manner. When the concentrations of propofol were 0.01,0.1,1,3,10,30,100,300,1000, and 3000μM, the inhibition rates of WT-hERG channel current were 3.8±2.4%, 13.9±7.9%,17.0±5.5%,23.2±5.0%,29.8±4.9%,43.7±5.4%,58.2±4.8%,66.4±7.5%, 71.2±4.3%, and 84.3±4.1%, respectively. The half maximal inhibitory concentration (IC50) of WT-hERG was 60.9±6.4μM.An "envelope of tails" protocol was used to investigate the time-dependence of propofol block. A significant block was achieved with a 50 ms depolarizing step after application of propofol. The extent of block was further increased with increasing pulse duration and maximal block was achieved at 1500 ms, demonstrating the inhibitory effect of propofol on WT-hERG current was time-dependent.The frequency dependence of WT-hERG current block was investigated by applying 30 repetitive pulses at 0.2 and 1 Hz. For control conditions, the HERG current amplitude during the pulse train decreased only slightly. Following exposure to 10μM propofol, application of the pulse train at either 0.2 or 1.0 Hz decreased current amplitude by 69.4±2.3% at 0.2 Hz and by 71.0±2.1% at 1 Hz (P>0.05). Thus, the effect of propofol on WT-hERG had no frequence-dependence.The block of WT-hERG by propofol had not altered the kinetic features of WT-hERG channel (activation, deactivation and inactivation).At 300μM concentration, propofol inhibited the WT-hERG channel by 66.3±7.5% while inhibited Y652A-hERG channel by 21.3±4.1%(P<0.05 vs WT-hERG). The IC50 value of Y652A-hERG was 2871.8±351.9μM. When the concentrations of propofol were 100,300,1000 and 3000μM, the inhibition rates of WT-hERG channel were 51.0±5.2%,62.4±5.9%,88.4±3.3%, and 94.7±2.1%, respectively, and the inhibition rates of F656C-hERG channel were 8.3±4.5%,21.2±6.3%,39.5±6.7%, and 45.0±8.3%, respectively (P<0.05 vs WT-hERG). Mutations in drug-binding sites (Y652A or F656C) of the hERG channel significantly attenuated the hERG current blockade by propofol.The results of Western blot and confocal imaging showed that propofol can not affect the protein trafficking of WT-hERG channel.Propofol blocked WT/Q738X-hERG channel in a concentration-dependent manner. When the concentrations of propofol were 0.1,1,3,10,30,100 and 1000μM, the inhibition of WT/Q738X-hERG were 2.9±4.9%,11.6±3.8%,33.7±3.2%,48.9±2.5%, 55.8±3.8%,80.1±2.9%, and 87.5±2.9%, respectively. The IC50 value of WT/Q738X-hERG was 14.2±2.8μM.The block of WT/Q738X-hERG by propofol had not altered the kinetic features of WT-hERG channel (activation, inactivation and deactivation).1. The Q738X mutation of hERG is nonfunctional and has no a dominant-negative effect on WT-hERG current function;2. Propofol inhibits WT-hERG channel in a concentration-and time-dependent manner, but not frequence-dependent; Propofol have not altered the kinetic features of WT-hERG channel (activation, deactivation, and inactivation); Mutations in drug-binding sites (Y652A or F656C) of the hERG channel attenuate the hERG current blockade by propofol; Propofol does not influence the protein trafficking of WT-hERG channel;3. Mutant Q738X can increase the inhibition of propofol on WT-hERG.