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Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming [alpha]-subunits that coassemble to form rapidly activating delayed rectifier [K.sup.+] current in the heart. LQT2-1inked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-1inked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-1inked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The [DELTA]Y475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue. human ether-a-go-go-related gene I; long QT syndrome 2; protein trafficking; E4031 doi: 10.1152/ajpheart.01236.2009.

KCNH2 (hERG1) encodes the [alpha]-subunit proteins for the rapidly activating delayed rectifier [K.sup.+] current ([I.sub.Kr]), a major [K.sup.+] current for cardiac myocyte repolarization. In isolated myocytes [I.sub.Kr] frequently is small in amplitude or absent, yet KCNH2 channels and [I.sub.Kr] are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and [I.sub.Kr] are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed [K.sup.+], [Na.sup.+], and L-type [Ca.sup.2+] channels, and in ventricular myoctyes [I.sub.Kr], slowly activating delayed rectifier [K.sup.+] current ([I.sub.Ks]), and inward rectifier [K.sup.+] current ([I.sub.1]), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current ([I.sub.KCNH2]) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished [I.sub.Kr]; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of [I.sub.KCNH2] and its mature channel protein over several hours. Thus the channel protein for [I.sub.KCNH2] and [I.sub.Kr] is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low [I.sub.Kr] density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. rapidly activating delayed rectifier potassium current; enzymes; myocyte isolation

Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels. Am J Physiol Heart Circ Physiol 286: H2434-H2441. 2004. First published February 19, 2004; 10.1152/ajpheart.00891.2003.--Long QT syndrome (LQTS) is a cardiac repolarization disorder that can lead to arrhythmias and sudden death. Chromosome 7-linked inherited LQTS (LQT2) is caused by mutations in human ether-a-go-go-related gene (HERG; KCNH2), whereas drug-induced LQTS is caused primarily by HERG channel block. Many common polymorphisms are functionally silent and have been traditionally regarded as benign and without physiological consequence. However, the identification of common nonsynonymous single nucleotide polymorphisms (nSNPs; i.e., amino-acid coding variants) with functional phenotypes in the SCN5A N[a.sup.+] channel and MiRP1 [K.sup.+] channel [beta]-subunit have challenged this viewpoint. In this report, we test the hypothesis that common missense HERG polymorphisms alter channel physiology. Comprehensive mutational analysis of HERG was performed on genomic DNA derived from a population-based cohort of sudden infant death syndrome and two reference allele cohorts derived from 100 African American and 100 Caucasian individuals. Amino acid-encoding variants were considered common polymorphisms if they were present in at least two of the three study cohorts with an allelic frequency >0.5%. Four nSNPs were identified: K897T, P967L, R1047L, and Q1068R. Wild-type (WT) and polymorphic channels were heterologously expressed in human embryonic kidney cells, and biochemical and voltage-clamp techniques were used to characterize their functional properties. All channel types were processed similarly, but several electrophysiological differences were identified: 1) K897T current density was lower than the other polymorphic channels; 2) K897T channels activated at more negative potentials than WT and R1047L; 3) K897T and Q1068R channels inactivated and recovered from inactivation faster than WT, P967L, and R1047L channels; and 4) K897T channels showed subtle differences compared with WT channels when stimulated with an action potential waveform. In contrast to K897T and Q1068R channels, P967L and R 1047L channels were electrophysiologically indistinguishable from WT channels. All HERG channels had similar sensitivity to block by cisapride. Therefore, some HERG polymorphic channels are electrophysiologically different from WT channels. ion channels; genetics; long QT syndrome; arrhythmia; sudden death

Drug-drug interactions pose a difficult drug safety problem, given the increasing number of individuals taking multiple medications and the relative complexity of assessing the potential for interactions. For example, sofosbuvir-based drug treatments have significantly advanced care for hepatitis C virus-infected patients, yet recent reports suggest interactions with amiodarone may cause severe symptomatic bradycardia and thus limit an otherwise extremely effective treatment. Here, we evaluated the ability of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to recapitulate the interaction between sofosbuvir and amiodarone in vitro, and more generally assessed the feasibility of hiPSC-CMs as a model system for drug-drug interactions. Sofosbuvir alone had negligible effects on cardiomyocyte electrophysiology, whereas the sofosbuvir-amiodarone combination produced dose-dependent effects beyond that of amiodarone alone. By comparison, GS-331007, the primary circulating metabolite of sofosbuvir, had no effect alone or in combination with amiodarone. Further mechanistic studies revealed that the sofosbuvir-amiodarone combination disrupted intracellular calcium (Ca2+) handling and cellular electrophysiology at pharmacologically relevant concentrations, and mechanical activity at supra-pharmacological (30x Cmax) concentrations. These effects were independent of the common mechanisms of direct ion channel block and P-glycoprotein activity. These results support hiPSC-CMs as a comprehensive, yet scalable model system for the identification and evaluation of cardioactive pharmacodynamic drug-drug interactions. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (INa), L-type calcium (ICa), hyperpolarization-activated pacemaker (If), transient outward potassium (Ito), inward rectifier potassium (IK1), and the rapidly and slowly activating components of delayed rectifier potassium (IKr and IKs, respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications. [ABSTRACT FROM AUTHOR]

Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming α-subunits that coassemble to form rapidly activating delayed rectifier K+ current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G6O1S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The ΔY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue. [ABSTRACT FROM AUTHOR]

β-Blockers are widely used in the treatment of cardiovascular diseases. However, their effects on HERG channels at comparable conditions remain to be defined. We investigated the direct acute effects of β-blockers on HERG current and the molecular basis of drug binding to HERG channels with mutations of putative common binding site (Y652A and F656C).β-Blockers were selected based on the receptor subtype. Wild-type, Y652A and F656C mutants of HERG channel were stably expressed in HEK293 cells, and the current was recorded by using whole-cell patch-clamp technique (23°C).Carvedilol (nonselective), propranolol (nonselective) and ICI 118551 (β2-selective) inhibited HERG current in a concentration-dependent manner (IC50 0.51, 3.9 and 9.2 μM, respectively). The IC50 value for carvedilol was a clinically relevant concentration. High metoprolol (β1-selective) concentrations were required for blockade (IC50 145 μM), and atenolol (β1-selective) did not inhibit the HERG current.Inhibition of HERG current by carvedilol, propranolol and ICI 118551 was partially but significantly attenuated in Y652A and F656C mutant channels. Affinities of metoprolol to Y652A and F656C mutant channels were not different compared with the wild-type.HERG current block by all β-blockers was not frequency-dependent.Drug affinities to HERG channels were different in β-blockers. Our results provide additional strategies for clinical usage of β-blockers. Atenolol and metoprolol may be preferable for patients with type 1 and 2 long QT syndrome. Carvedilol has a class III antiarrhythmic effect, which may provide the rationale for a favourable clinical outcome compared with other β-blockers as suggested in the recent COMET (Carvedilol Or Metoprolol European Trial) substudy.British Journal of Pharmacology (2006) 147, 642–652. doi:10.1038/sj.bjp.0706508; published online 28 November 2005 [ABSTRACT FROM AUTHOR]

KCNH2 (hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current (IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current (IKs), and inward rectifier K+ current (IKl), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCN2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. [ABSTRACT FROM AUTHOR]

KCNH2 (hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current (IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current (IKs), and inward rectifier K+ current (IKl), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCN2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. [ABSTRACT FROM AUTHOR]