Our previous study found that Ca2+/calmodulin-dependent protein kinase II (CaMKII) potentiates the slow delayed rectifier K+ current (I-Ks) in sinoatrial node (SAN) pacemaker cells. Recently, oxidative activation of CaMKII has emerged as a major cause of SAN dysfunction; however, its correlation with I-Ks regulation remains unclear. In this study, we investigated the effect of hydrogen peroxide (H2O2) on I-Ks in SAN cells isolated from guinea pig heart. Whole-cell patch-clamp recordings were performed using an EGTA (5 mM) pipette solution to stabilize intracellular Ca2+ levels (pCa 7). The results showed that 5 min of H2O2 (100 mu M) perfusion initiated an increase in I-Ks, which gradually increased to saturation (similar to 60.5 % enhancement from baseline to saturation) after 10 min of H2O2 exposure. In contrast, IKs remained almost unchanged in the presence of catalase (1000 units mL- 1). These observations were replicable in atrial and ventricular cardiomyocytes. H2O2 failed to stimulate KCNQ1/KCNE1 currents in HEK and CHO cells expressing low CaMKII levels. In SAN cells, H2O2-induced IKs enhancement was strongly attenuated by intracellular dialysis with a lower Ca2+ concentration (pCa 10) or by pretreatment with KN-93 (1 mu M), suggesting that Ca2+/calmodulin binding to CaMKII is a prerequisite for CaMKII activation. Autocamtide-2 inhibitory peptide (AIP, 1 mu M), an inhibitor of the catalytic domain of CaMKII, almost completely abolished the H2O2-induced potentiation of I-Ks. Taken together, these findings imply that H2O2 enhances cardiac I-Ks through the oxidative activation of CaMKII.

Propofol is a broadly used intravenous anesthetic agent that can cause cardiovascular effects, including bradycardia and asystole. A possible mechanism for these effects is slowing cardiac pacemaker activity due to inhibition of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. However, it remains unclear how propofol affects the allosteric nature of the voltage- and cAMP-dependent gating mechanism in HCN channels. To address this aim, we investigated the effect of propofol on HCN channels (HCN4 and HCN2) in heterologous expression systems using a whole-cell patch clamp technique. The extracellular application of propofol substantially suppressed the maximum current at clinical concentrations. This was accompanied by a hyperpolarizing shift in the voltage dependence of channel opening. These effects were significantly attenuated by intracellular loading of cAMP, even after considering the current modification by cAMP in opposite directions. The differential degree of propofol effects in the presence and absence of cAMP was rationalized by an allosteric gating model for HCN channels, where we assumed that propofol affects allosteric couplings between the pore, voltage-sensor, and cyclic nucleotide-binding domain (CNBD). The model predicted that propofol enhanced autoinhibition of pore opening by unliganded CNBD, which was relieved by the activation of CNBD by cAMP. Taken together, these findings reveal that propofol acts as an allosteric modulator of cAMP-dependent gating in HCN channels, which may help us to better understand the clinical action of this anesthetic drug.

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I Kur). Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as an effective treatment of re-entrant based atrial fibrillation, because Kv1.5 is highly expressed in human cardiac atria but scarcely in ventricles. The Kv1.5 blockade is also expected to be used in cancer therapeutics since Kv1.5 is overexpressed in some types of human tumors. Here, we investigated the blockade of hKv1.5 channels by HMQ1611, a symmetrical biphenyl derivative. hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. The effects of HMQ1611 on wild-type and 13 hKv1.5 mutant channels were examined using the whole-cell patch-clamp method, and molecular docking simulation was conducted to predict the docking position of HMQ1611 within Kv1.5 channels. We showed that HMQ1611 reversibly inhibited the hKv1.5 current in a concentration-dependent manner (IC50 = 2.07 μM). HMQ1611 blockade of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. HMQ1611 inhibition was significantly reduced in the T479A, T480A, V505A, I508A, L510A, V512A, and V516A hKv1.5 mutant channels. Molecular docking analysis predicted that V505, V512, and T480 were involved in the blocking action of HMQ1611 on hKv1.5 channels. These results suggest that HMQ1611 inhibits hKv1.5 currents as an open channel blocker. Amino acid residues located at the base of the selectivity filter (T479 and T480) and in the S6 segment (V505, I508, L510, V512, and V516) of hKv1.5 appear to constitute potential binding sites for HMQ1611.

Background and Purpose The slowly activating delayed rectifier K+ channel (I-Ks), composed of pore-forming KCNQ1 alpha-subunits and ancillary KCNE1 beta-subunits, regulates ventricular repolarization in human heart. Propofol, at clinically used concentrations, modestly inhibits the intact (wild-type) I-Ks channels and is therefore unlikely to appreciably prolong QT interval in ECG during anaesthesia. However, little information is available concerning the inhibitory effect of propofol on I-Ks channel associated with its gene variants implicated in QT prolongation. The KCNE1 single nucleotide polymorphism leading to D85N is associated with drug-induced QT prolongation and therefore regarded as a clinically important genetic variant. This study examined whether KCNE1-D85N affects the sensitivity of I-Ks to inhibition by propofol. Experimental Approach Whole-cell patch-clamp and immunostaining experiments were conducted in HEK293 cells and/or mouse cardiomyocyte-derived HL-1 cells, transfected with wild-type KCNQ1, wild-type or variant KCNE1 cDNAs. Key Results Propofol inhibited KCNQ1/KCNE1-D85N current more potently than KCNQ1/KCNE1 current in HEK293 cells and HL-1 cells. Immunostaining experiments in HEK293 cells revealed that pretreatment with propofol (10 mu M) did not appreciably affect cell membrane expression of KCNQ1 and KCNE1 proteins in KCNQ1/KCNE1 and KCNQ1/KCNE1-D85N channels. Conclusion and Implications The KCNE1 polymorphism D85N significantly elevates the sensitivity of I-Ks to inhibition by propofol. This study detects a functionally important role of KCNE1-D85N polymorphism in conferring genetic susceptibility to propofol-induced QT prolongation and further suggests the possibility that the inhibitory action of anaesthetics on ionic currents becomes exaggerated in patients carrying variants in genes encoding ion channels.

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both β1- and β2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β2-adrenoceptor stimulation than β1-adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

The atrioventricular (AV) node is the only conduction pathway where electrical impulse can pass from atria to ventricles and exhibits spontaneous automaticity. This study examined the function of the rapid- and slow-activating delayed rectifier K + currents ( I Kr and I Ks ) in the regulation of AV node automaticity. Isolated AV node cells from guinea pigs were current- and voltage-clamped to record the action potentials and the I Kr and I Ks current. The expression of I Kr or I Ks was confirmed in the AV node cells by immunocytochemistry, and the positive signals of both channels were localized mainly on the cell membrane. The basal spontaneous automaticity was equally reduced by E4031 and HMR-1556, selective blockers of I Kr and I Ks , respectively. The nonselective β-adrenoceptor agonist isoproterenol markedly increased the firing rate of action potentials. In the presence of isoproterenol, the firing rate of action potentials was more effectively reduced by the I Ks inhibitor HMR-1556 than by the I Kr inhibitor E4031. Both E4031 and HMR-1556 prolonged the action potential duration and depolarized the maximum diastolic potential under basal and β-adrenoceptor–stimulated conditions. I Kr was not significantly influenced by β-adrenoceptor stimulation, but I Ks was concentration-dependently enhanced by isoproterenol (EC 50 : 15 nM), with a significant negative voltage shift in the channel activation. These findings suggest that both the I Kr and I Ks channels might exert similar effects on regulating the repolarization process of AV node action potentials under basal conditions; however, when the β-adrenoceptor is activated, I Ks modulation may become more important.

Pregnancy causes changes in the uterus, such as increased cell volume and altered water content. However, the mechanisms that protect the structure and maintain the function of uterine smooth muscle cells against these changes during pregnancy have not been clarified. This study focused on the volume-regulated anion channel (VRAC), which opens with cell swelling under low osmotic pressure and releases Cl- ions and various organic osmolytes to resist cell swelling and regulates a wide range of biological processes such as cell death. In this study, myometrial smooth muscle (MSM) tissues and cells (MSMCs) were collected from non-pregnant and pregnant mice. Using western blotting and immunocytochemistry, leucine-rich repeat containing protein 8A (LRRC8A), an essential membrane protein that constitutes part of the VRAC, was determined to be diffused throughout MSMCs including in the cell membrane. Patch-clamp experiments were performed to investigate the electrophysiology of swelling-induced Cl- currents (ICl, swell) mediated by the VRAC. No significant changes between non-pregnancy and pregnancy groups were observed in either the expression density of LRRC8A or the current density of ICl, swell, however the presence of LRRC8A on the cell membrane was significantly increased in the third trimester of pregnancy compared to the non-pregnancy. This study suggests that the VRAC may play a role, such as maintaining cellular homeostasis in the pregnant MSM.

Dexmedetomidine (DEX), an alpha(2)-adrenoreceptor (alpha(2)-AR) and imidazoline receptor agonist, is most often used for the sedation of patients in the intensive care unit. Its administration is associated with an increased incidence of bradycardia; however, the precise mechanism of DEX-induced bradycardia has yet to be fully elucidated. This study was undertaken to examine whether DEX modifies pacemaker activity and the underlying ionic channel function through alpha(2)-AR and imidazoline receptors. The whole-cell patch-clamp techniques were used to record action potentials and related ionic currents of sinoatrial node cells in guinea pigs. DEX (>= 10 nM) reduced sinoatrial node automaticity and the diastolic depolarization rate. DEX reduced the amplitude of hyperpolarization-activated cation current (I-f or I-h) the pacemaker current, even within the physiological pacemaker potential range. DEX slowed the If current activation kinetics and caused a significant shift in the voltage dependence of channel activation to negative potentials. In addition, efaroxan, an alpha(2)-AR and imidazoline I-1 receptor antagonist, attenuated the inhibitory effects of DEX on sinoatrial node automaticity and If current activity, whereas yohimbine, an alpha(2)-AR-selective antagonist, did not. DEX did not affect the current activities of other channels, including rapidly and slowly activating delayed rectifier K+ currents (I-Kr and I-Ks), L-type Ca2+ current (I-Ca,I-L), Na+/Ca2+ exchange current (I-NCX), and muscarinic K+ current (I-K,I-ACh). Our results indicate that DEX, at clinically relevant concentrations, induced a negative chronotropic effect on the sinoatrial node function through the downregulation of I-f current through an imidazoline I-1 receptor other than the alpha(2)-AR in the clinical setting.

Background and Purpose Volatile anaesthetics have been shown to differentially modulate mammalianShaker-related voltage-gated potassium (K(v)1.x) channels. This study was designed to investigate molecular and cellular mechanisms underlying the modulatory effects of desflurane or sevoflurane on human K(v)1.5 (hK(v)1.5) channels. Experimental Approach Thirteen single-point mutations were constructed within pore domain of hK(v)1.5 channel using site-directed mutagenesis. The effects of desflurane or sevoflurane on heterologously expressed wild-type and mutant hK(v)1.5 channels were examined by whole-cell patch-clamp technique. A computer simulation was conducted to predict the docking pose of desflurane or sevoflurane within hK(v)1.5 channel. Key Results Both desflurane and sevoflurane increased hK(v)1.5 current at mild depolarizations but decreased it at strong depolarizations, indicating that these anaesthetics produce both stimulatory and inhibitory actions on hK(v)1.5 channels. The inhibitory effect of desflurane or sevoflurane on hK(v)1.5 channels arose primarily from its open-channel blocking action. The inhibitory action of desflurane or sevoflurane on hK(v)1.5 channels was significantly attenuated in T480A, V505A, and I508A mutant channels, compared with wild-type channel. Computational docking simulation predicted that desflurane or sevoflurane resides within the inner cavity of channel pore and has contact with Thr479, Thr480, Val505, and Ile508. Conclusion and Implications Desflurane and sevoflurane exert an open-channel blocking action on hK(v)1.5 channels by functionally interacting with specific amino acids located within the channel pore. This study thus identifies a novel molecular basis mediating inhibitory modulation of hK(v)1.5 channels by desflurane and sevoflurane.

Abstract Background Early repolarization syndrome (ERS) is characterized by J-point elevation in the ECG and ventricular fibrillation (VF). Several mutations in genes encoding cardiac ion channels have been reported as the causes for ERS. For the treatment of ERS, clinical studies have shown that quinidine is effective for the suppression of electrical storm. However, the mechanism or the optimal concentration for quinidine to suppress the electrical storm has not been elucidated yet. Purpose The aim of the present study is to clarify pharmacological effect of quinidine on mutant Kv4.3 channel by electrophysiological analysis and to establish theoretically effective treatment for ERS. Methods A KCND3 mutation, p.G306A, identified heterozygously in a 12-year-old boy was examined by whole-cell patch-clamp methods using CHO cells. We performed functional analysis of the Kv4.3 channels encoded by KCND3 of wild-type (WT), heterozygous (WT/G306A), or homozygous (G306A) mutants. Pharmacological normalizing effects of quinidine to the WT and mutant channels were investigated by loading test in various concentration. The sensitivity of quinidine was evaluated in terms of the concentration of the clinical course and the loading test. Results The patient suffered VF while sleeping or under sedation. Significant J-point elevations in multiple leads were recorded and he was diagnosed as ERS. Quinidine administration in the serum concentration of 1.2 to 3.1 μM was effective to stop his VF storm. Mutant Kv4.3 currents showed significantly slow inactivation time course (Fig 1), which meant that the mutation caused the gain-of-function channel. Quinidine loading to the mutant Kv4.3 normalized the inactivation time course in concentration-dependent manner (Fig 2, 3). In the loading concentration of 1 μM, the peak currents were not changed regardless of the voltage (Fig 4). The optimal serum concentration of quinidine in clinical use as multi-ion-channels blocker is 6 to 15 μM and the concentration was higher than that of the patient to stop the VF storm. The electrophysiological analysis showed that the low dose loading of quinidine was effective to prevent the gain-of function change of mutant Kv4.3 channels. Electrophysiological analysis Conclusions We showed the pharmacological mechanism of quinidine against the mutant Kv4.3 channels identified in an ERS patient. Even the low concentration of quinidine was effective to normalize the slow inactivation, gain-of-function, in mutant Kv4.3 channels. Our data would be helpful to confirm the optimal concentration of quinidine for ERS patients to avoid adverse effect.

[This corrects the article DOI: 10.3892/etm.2018.6422.].

BACKGROUND/AIMSThe phenylalkylamine class of L-type Ca2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium.METHODSSite-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore.RESULTSVerapamil preferentially blocked hKv1.5 channel in its open state with IC50 of 2.4±0.6 μM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516.CONCLUSIONVerapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans.

BACKGROUND:Missense mutations in KCNH2, a gene encoding the Kv11.1 channel, cause long QT syndrome (LQTS) type 2 primarily by disrupting the intracellular transport of Kv11.1 to the plasma membrane. The present study aimed to clarify the functional changes by two novel KCNH2 missense mutations.METHODS:We performed genetic screening of three unrelated symptomatic LQTS probands with family histories of cardiac symptoms. Chinese hamster ovary cells were transfected with wild-type (WT) and/or mutant KCNH2 plasmid and examined by patch-clamp technique. Immunostaining and confocal microscopy were performed to evaluate the intracellular localization of WT and homozygous mutant Kv11.1 in human embryonic kidney cells. For the study of trafficking rescue, we used low-temperature incubation (30°C). We also examined pharmacological rescue of homozygous mutant Kv11.1 current in cells treated with E-4031 or dofetilide.RESULTS:We identified two novel KCNH2 missense mutations, G785D and T826I. Electrophysiological study showed that both mutant channels were nonfunctional in homozygous condition and reduced current densities by half in heterozygous condition compared with WT Kv11.1. Heterozygous Kv11.1-G785D produced a significant positive shift in activation and a significant negative shift in inactivation, whereas heterozygous Kv11.1-T826I caused no kinetic changes. Immunostaining revealed that both were transport-refractory mutations. Incubation at 30°C rescued plasma membrane expression of Kv11.1-T826I but not G785D. We confirmed low-temperature-induced restoration of homozygous Kv11.1-T826I transport by functional current measurements. In contrast, incubation with E-4031 or dofetilide failed to produce measurable currents in both homozygous mutant channels.CONCLUSIONS:Two novel KCNH2 mutations disrupted the intracellular transport of Kv11.1. Low-temperature incubation rescued plasma membrane expression of Kv11.1-T826I but not G785D. Both mutations exerted loss-of-function effects on Kv11.1 and explained the phenotypes of the mutation carriers.

The present study has demonstrated that P-5m octapeptide (P-5m) has therapeutic potential in metastatic human hepatocarcinoma, possibly through the modulation of matrix metalloproteinase-2 expression. The purpose of the present study was to evaluate the antitumor effect of P-5m combined with 5-fluorouracil (5-Fu) on the treatment of hepatoma 22 (H22) hepatocarcinoma malignant ascites in a mouse model. The inhibitory effect on the growth of mouse ascites tumors was monitored by measuring body weight gain, survival time, ascites volume, numbers of tumor cells, DNA synthesis and peritoneal capillary permeability analysis. The present data revealed a significant reduction in ascites volume and cell count in mice that were treated with P-5m plus 5-Fu. Furthermore, the median survival time in mice in the combination group was prolonged compared with the disease control group. Moreover, a significant reduction in the total H22 ascites cell count in mice from the combination group was observed when compared with the disease control group. P-5m plus 5-Fu was able to induce the cell cycle arrest and inhibit the peritoneal capillary permeability of the mice. To conclude, the present study indicated that P-5m may have therapeutic potential in ascites caused by hepatocellular carcinoma.

Abstract: Reperfusion of ischemic myocardium is accompanied by intracellular Ca2+ overload, leading to cardiac dysfunction. However, the mechanisms underlying intracellular Ca2+ overload have yet to be fully elucidated. The mechanism may involve the activation of store-operated Ca2+ entry, which is primarily mediated through the transient receptor potential canonical (TRPC) channels. This study was undertaken to examine the possible involvement of TRPC channels in the development of contractile dysfunction associated with reperfusion of ischemic myocardium using a mouse heart model. The functional expression of TRPC channels was confirmed in mouse ventricular myocytes using immunocytochemistry, Western blotting, and patch-clamp experiments. The left ventricular functions were assessed by measuring left ventricular end-diastolic pressure, left ventricular developed pressure, and its first derivatives in a Langendorff-perfused mouse heart subjected to 30 minutes of normothermic (37°C) global ischemia followed by 60 minutes of reperfusion. Under control conditions, left ventricular functions were deteriorated during reperfusion, which was significantly ameliorated by administration of the TRPC channel blockers 2-aminoethoxydiphenyl borate and La3+ during initial 5 minutes of reperfusion. Our findings suggest that TRPC channels are involved in mediating contractile dysfunction during reperfusion of ischemic myocardium and detect TRPC channels as a potential therapeutic target for preventing myocardial ischemia/reperfusion injury.

Congenital long QT syndrome (LQTS) caused by compound mutations is usually associated with more severe clinical phenotypes, however, the underlying mechanisms remain unclear. We identified a LQTS family harboring three compound mutations in different genes ( KCNQ1 -R174C, KCNH2 -E1039X and SCN5A -

Dr. Wei-Guang Ding’s given name and family name were inadvertently interchanged initially. The correct names are as shown above.

BackgroundIn the clinical setting, verapamil is contraindicated in neonates and infants, because of the perceived risk of hypotension or bradyarrhythmia. However, it remains unclear whether there is an age-dependent difference in the sensitivity of cardiac L-type Ca2+ channel current (ICa,L) to inhibition by verapamil.MethodsVentricular myocytes were enzymatically dissociated from the hearts of six different age groups (0, 7, 14, 21, 28 days, and 10-15 weeks) of mice, using a similar Langendorff-perfusion method. Whole-cell patch-clamp technique was applied to examine the sensitivity of ICa,L to inhibition, by three classes of structurally different L-type Ca2+ channel antagonists.ResultsVerapamil, nifedipine, and diltiazem concentration-dependently blocked the ventricular ICa,L in all six age groups. However, although nifedipine and diltiazem blocked ventricular ICa,L with a similar potency in all age groups, verapamil more potently blocked ventricular ICa,L in day 0, day 7, day 14, and day 21 mice, than in day 28, and 10-15-week mice.ConclusionIn a mouse heart model, ventricular ICa,L before the weaning age (~21 days of age) exhibited a higher sensitivity to inhibition by verapamil than that after the weaning age, which may explain one possible mechanism associated with the development of verapamil-induced hypotension in human neonates and infants.

Congenital long QT syndrome (LQTS) caused by compound mutations is usually associated with more severe clinical phenotypes. We identified a LQTS family harboring three compound mutations in different genes ( KCNQ1 -R174C, hERG -E1039X and SCN5A -E428K). KCNQ1 -R174C, hERG -E1039X and SCN5A -E428K mutations and/or relevant wild-type (WT) cDNAs were respectively expressed in mammalian cells. I Ks -like, I Kr -like, I Na -like currents and the functional interaction between KCNQ1-R174C and hERG-E1039X channels were studied using patch-clamp and immunocytochemistry techniques. (1) Expression of KCNQ1-R174C alone showed no I Ks . Co-expression of KCNQ1-WT + KCNQ1-R174C caused a loss-of-function in I Ks and blunted the activation of I Ks in response to isoproterenol. (2) Expression of hERG-E1039X alone and co-expression of hERG-WT + hERG-E1039X negatively shifted inactivation curves and decelerated the recovery time from inactivation. (3) Expression of SCN5A-E428K increased peak I Na , but had no effect on late I Na . (4) I Ks and I Kr interact, and hERG -E1039X caused a loss-of-function in I Ks . (5) Immunocytochemical studies indicated that KCNQ1-R174C is trafficking defective and hERG-E1039X is defective in biosynthesis/degradation, but the abnormities were rescued by co-expression with WT. Thus, KCNQ1 -R174C and hERG -E1039X disrupted I Ks and I Kr functions, respectively. The synergistic lesion, caused by KCNQ1 -R174C and hERG -E1039X in I Ks , is very likely why patients showed more severe phenotypes in the compound mutation case.