Background— The KCNH2 or human ether-a-go-go related gene ( hERG ) encodes the Kv11.1 α-subunit of the rapidly activating delayed rectifier K + current ( I Kr ) in the heart. Type 2 congenital long-QT syndrome (LQT2) results from KCNH2 mutations that cause loss of Kv11.1 channel function. Several mechanisms have been identified, including disruption of Kv11.1 channel synthesis (class 1), protein trafficking (class 2), gating (class 3), or permeation (class 4). For a few class 2 LQT2-Kv11.1 channels, it is possible to increase surface membrane expression of Kv11.1 current ( I Kv11.1 ). We tested the hypotheses that (1) most LQT2 missense mutations generate trafficking-deficient Kv11.1 channels, and (2) their trafficking-deficient phenotype can be corrected. Methods and Results— Wild-type (WT)-Kv11.1 channels and 34 missense LQT2-Kv11.1 channels were expressed in HEK293 cells. With Western blot analyses, 28 LQT2-Kv11.1 channels had a trafficking-deficient (class 2) phenotype. For the majority of these mutations, the class 2 phenotype could be corrected when cells were incubated for 24 hours at reduced temperature (27°C) or in the drugs E4031 or thapsigargin. Four of the 6 LQT2-Kv11.1 channels that had a wild-type–like trafficking phenotype did not cause loss of Kv11.1 function, which suggests that these channels are uncommon sequence variants. Conclusions— This is the first study to identify a dominant mechanism, class 2, for the loss of Kv11.1 channel function in LQT2 and to report that the class 2 phenotype for many of these mutant channels can be corrected. This suggests that if therapeutic strategies to correct protein trafficking abnormalities can be developed, it may offer clinical benefits for LQT2 patients.

It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal C-terminus. Also, most pore mutations—in contrast to intracellular domain mutations —were found to have severe dominant-negative effects when co-expressed with wild type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild type subunits to form heteromeric channels.

The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop. Bmal1 encodes a core molecular clock transcription factor. Germline Bmal1 knockout mice show a loss of circadian variation in heart rate and blood pressure, and they develop dilated cardiomyopathy. We tested the role of the molecular clock in adult cardiomyocytes by generating mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCS⌬Bmal1). ECG telemetry showed that cardiomyocyte-specific deletion of Bmal1 (iCS⌬Bmal1 Ϫ/Ϫ ) in adult mice slowed heart rate, prolonged RR and QRS intervals, and increased episodes of arrhythmia. Moreover, isolated iCS⌬Bmal1 Ϫ/Ϫ hearts were more susceptible to arrhythmia during electromechanical stimulation. Examination of candidate cardiac ion channel genes showed that Scn5a, which encodes the principle cardiac voltage-gated Na ϩ channel (NaV1.5), was circadianly expressed in control mouse and rat hearts but not in iCS⌬Bmal1 Ϫ/Ϫ hearts. In vitro studies confirmed circadian expression of a human Scn5a promoter-luciferase reporter construct and determined that overexpression of clock factors transactivated the Scn5a promoter. Loss of Scn5a circadian expression in iCS⌬Bmal1 Ϫ/Ϫ hearts was associated with decreased levels of NaV1.5 and Na ϩ current in ventricular myocytes. We conclude that disruption of the molecular clock in the adult heart slows heart rate, increases arrhythmias, and decreases the functional expression of Scn5a. These findings suggest a potential link between environmental factors that alter the cardiomyocyte molecular clock and factors that influence arrhythmia susceptibility in humans. cardiac excitability; circadian; heart; ion channels; Scn5a; Na ϩ current CIRCADIAN RHYTHMS are approximate 24-h cycles in biology. These rhythms are present at the systems level, the tissue level, the single cell and molecular levels (16,33,34,38). There are several examples of circadian rhythms in the cardiovascular system, with heart rate, blood pressure, and substrate metabolism exhibiting distinct oscillations over time of day (12,13,40,41). The mechanism that underlies circadian function is the molecular clock. The molecular clock is defined, in a simple way, by a transcription-translation feedback mechanism that is composed of the core clock genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2. CLOCK and BMAL1 are transcription factors that heterodimerize and activate transcription of Per1, Per2, Cry1, and Cry2. PER1, PER2, CRY1, and CRY2 form multimers in the cytoplasm of the cell, translocate to the nucleus, and act to inhibit CLOCK:BMAL1 function. This cycle takes ϳ24 h and is the fundamental mechanism underlying circadian rhythms. Components of the core clock have also been shown to regulate the expression of genes outside the clock mechanism, and these genes are designated as clock-controlled genes (CCGs). CCGs often encode transcription factors or proteins that control rate-limiting steps in cell physiology (42).In the last 8 years, resear...

No abstract

Background and purpose: Fluoxetine (Prozac s ) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. Experimental approach: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K þ and SCN5A Na þ channels heterologously expressed in HEK293 cells. Key results: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (I hERG ). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of I hERG but did not alter fluoxetine's effect on hERG channel protein trafficking. Conclusions and implications: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce I hERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

No abstract

Multiple Ca2+ channel beta-subunit (Ca(v)beta) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Ca(v)beta subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Ca(v)beta genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Ca(v)beta isoforms. A total of 18 different Ca(v)beta isoforms were detected in both canine and human ventricles including splice variants from all four Ca(v)beta genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Ca(v)beta subunit genes are expressed at the protein level, and the Ca(v)beta subunits show differential subcellular localization with Ca(v)beta1b, Ca(v)beta2, and Ca(v)beta3 predominantly localized to the T-tubule sarcolemma, whereas Ca(v)beta1a and Ca(v)beta4 are more prevalent in the surface sarcolemma. Coexpression of the novel Ca(v)beta2c subunits (Ca(v)beta(2cN1), Ca(v)beta(2cN2), Ca(v)beta(2cN4)) with the pore-forming alpha1C (Ca(v)1.2) and Ca(v)alpha2delta subunits in HEK 293 cells resulted in a marked increase in ionic current and Ca(v)beta2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Ca(v)beta subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.

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 Na+ channel and MiRP1 K+ channel β-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 R1047L 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.