Kinetics of drug interaction with the Kv11.1 potassium channel.

The Kv11.1 potassium channel is the molecular target for the majority of drugs implicated in acquired long QT syndrome, the most common cause of drug-induced sudden cardiac death, and a common reason for drug restriction or withdrawal from the market. While the IC50 for block of Kv11.1 is commonly used to estimate the risk of acquired long QT syndrome, this approach is crude, and it is widely accepted that the kinetics of drug interactions with the channel are a critical component in understanding their mechanism of action and risk profiles. In this study we report the first directly measured kinetics of block and unblock of Kv11.1 by a QT prolonging drug: the antipsychotic clozapine. Our data show that clozapine binding to Kv11.1 is complex. There are at least two kinetically distinct components to both block and unblock, while the kinetics of unblock are dependent on the dose or duration of drug application. Based on these observations, we have proposed a model incorporating kinetically distinct binding to the open and inactivated states of Kv11.1 that can describe the observed kinetic features of clozapine block and correctly predict the overall affinity and apparent nonstate-dependent interaction of clozapine with Kv11.1. Mechanistic insights into drug block of Kv11.1 gained though detailed kinetic analyses such as this have a potential role in development of drugs targeted to specific channel states to reduce unwanted side effects, as well as in the design of better high-throughput preclinical tests for assessing the proarrhythmic effects of QT prolonging drugs.

Not all hERG pore domain mutations have a severe phenotype: G584S has an inactivation gating defect with mild phenotype compared to G572S, which has a dominant negative trafficking defect and a severe phenotype.

INTRODUCTION: Mutations in the pore domain of the human ether-a-go-go-related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification. METHODS AND RESULTS: We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584S-hERG channels. CONCLUSIONS: hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes; G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.

Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels.

Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.

Off-label use of medicines: consensus recommendations for evaluating appropriateness.

Off-label prescribing is the prescription of a registered medicine for a use that is not included in the product information. The practice is common, with rates up to 40% in adults and up to 90% in paediatric patients. Off-label prescribing is not illegal and may sometimes be clinically appropriate, but is associated with a number of clinical, safety and ethical issues. To date, no explicit guidance has been available to help clinicians assess appropriateness in off-label prescribing. We describe the development of a guide for clinicians, policymakers and funders of health care in evaluating the appropriateness of medicines proposed for off-label use. Three broad categories of appropriate off-label use are identified:off-label use justified by high-quality evidence; use within the context of a formal research proposal; and exceptional use, justified by individual clinical circumstances. An appropriate process for informed consent is proposed for each category. If there is no high-quality evidence supporting off-label use, and the medicine is not suitable for exceptional or research indications, its use is generally not recommended. This will reduce inappropriate use, enhance patient safety by reducing exposure to unnecessary risk, and may stimulate more clinically relevant medicines research.

Tryptophan scanning mutagenesis of the HERG K+ channel: the S4 domain is loosely packed and likely to be lipid exposed.

Inherited mutations or drug-induced block of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death. The fourth transmembrane domain (S4) of these channels contains multiple positive charges that move across the membrane electric field in response to changes in transmembrane voltage. In HERG K+ channels, the movement of the S4 domain across the transmembrane electric field is particularly slow. To examine the basis of the slow movement of the HERG S4 domain and specifically to probe the relationship between the S4 domain with the lipid bilayer and rest of the channel protein, we individually mutated each of the S4 amino acids in HERG (L524-L539) to tryptophan, and characterized the activation and deactivation properties of the mutant channels in Xenopus oocytes, using two-electrode voltage-clamp methods. Tryptophan has a large bulky hydrophobic sidechain and so should be tolerated at positions that interact with lipid, but not at positions involved in close protein-protein interactions. Significantly, we found that all S4 tryptophan mutants were functional. These data indicate that the S4 domain is loosely packed within the rest of the voltage sensor domain and is likely to be lipid exposed. Further, we identified residues K525, R528 and K538 as being the most important for slow activation of the channels.

Molecular basis of slow activation of the human ether-a-go-go related gene potassium channel.

The human ether-á-go-go related gene (HERG) encodes the pore forming alpha-subunit of the rapid delayed rectifier K(+) channel which is central to the repolarization phase of the cardiac action potential. HERG K(+) channels have unusual kinetics characterized by slow activation and deactivation, yet rapid inactivation. The fourth transmembrane domain (S4) of HERG, like other voltage-gated K(+) channels, contains multiple positive charges and is the voltage sensor for activation. In this study, we mutated each of the positively charged residues in this region to glutamine (Q), expressed the mutant and wild-type (WT) channels in Xenopus laevis oocytes and studied them using two-electrode voltage clamp methods. K525Q channels activated at more hyperpolarized potentials than WT, whereas all the other mutant channels activated at more depolarized potentials. All mutants except for R531Q also had a reduction in apparent gating charge associated with activation. Mutation of K525 to cysteine (C) resulted in a less dramatic phenotype than K525Q. The addition of the positively charged MTSET to K525C altered the phenotype to one more similar to K525Q than to WT. Therefore it is not charge per se, but the specific lysine side chain at position 525, that is crucial for stabilizing the closed state. When rates of activation and deactivation for WT and mutant channels were compared at equivalent total (chemical + electrostatic) driving forces, K525Q and R528Q accelerated activation but had no effect on deactivation, R531Q slowed activation and deactivation, R534Q accelerated activation but slowed deactivation and R537Q accelerated deactivation but had no effect on activation. The main conclusions we can draw from these data are that in WT channels K525 stabilizes the closed state, R531 stabilizes the open state and R534 participates in interactions that stabilize pre-open closed states.

Effect of pravastatin compared with placebo initiated within 24 hours of onset of acute myocardial infarction or unstable angina: the Pravastatin in Acute Coronary Treatment (PACT) trial.

BACKGROUND: The efficacy of statin drugs after an acute coronary event is now well established, but the evidence for statin use in the early treatment of acute coronary events remains unclear. METHODS: We tested the effects of administering pravastatin within 24 hours of the onset of symptoms in patients with unstable angina, non-ST-segment elevation myocardial infarction, or ST-segment elevation myocardial infarction. Patient recruitment of 10,000 with 1200 end points was planned, but the trial was stopped early. A total of 3408 patients were randomly assigned to treatment with pravastatin (1710 patients) or matching placebo (1698 patients). Treatment was continued for 4 weeks. The primary end point of the study was a composite of death, recurrence of myocardial infarction, or readmission to hospital for unstable angina within 30 days of random assignment. RESULTS: The primary end point occurred in 199 of patients allocated to pravastatin (11.6%) and in 211 patients allocated to placebo (12.4%). A relative risk reduction of 6.4% favored allocation to pravastatin but was not statistically significant (95% CI, -13.2% to 27.6%). No adverse effects were seen. CONCLUSIONS: We conclude that 20 to 40 mg of pravastatin can be safely administered within 24 hours of the onset of symptoms of an acute coronary event, with a favorable but not significant trend in outcome at 30 days compared with placebo.

Inherited cardiac arrhythmia syndromes: what have they taught us about arrhythmias and anti-arrhythmic therapy?

1. In recent years, the identification of the gene defects in a vast array of monogenic disorders has revolutionized our understanding of the basic mechanisms underlying numerous disease processes. 2. Mutations in cardiac ion channels have been identified as the basis of a wide range of inherited arrhythmia syndromes, including the congenital long QT syndromes, Brugada syndrome, Lenegre syndrome, Andersen's disease and familial atrial fibrillation. 3. Identification of mutations in the human-ether-a-go-go-related gene (HERG) K(+) channel as the molecular basis of congenital long QT syndrome type 2 also led to the discovery that HERG is the molecular target for the vast majority of drugs (both cardiac and non-cardiac) that cause drug-induced arrhythmias. This has had profound implications not only for the development of anti-arrhythmic agents, but also for drug development in general. 4. The sequencing of the human genome in a sense represents the pinnacle of the reductionist era of molecular medicine. The great challenge now is to re-integrate the information gathered during the 'reductionist era' to provide a better understanding of the intact organism. Computer modelling is likely to be a key component of that re-integration process.

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition.

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.

The HERG K+ channel: progress in understanding the molecular basis of its unusual gating kinetics.

The HERG K+ channel has very unusual kinetic behaviour that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarisation as well as in preventing lethal ventricular arrhythmias. Extensive mutagenesis of the HERG K+ channel has allowed identification of which regions of the channel are important for the unusual kinetic behaviour of the channel. Furthermore, structural studies on scorpion toxins that potently inhibit HERG are beginning to provide clues as to the structural differences between HERG and other voltage-gated K+ channels.

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation.

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.

Digoxin in heart failure and cardiac arrhythmias.

HEART FAILURE: Digoxin therapy has no effect on mortality in heart failure. Digoxin may be useful for maintaining clinical stability and exercise capacity in patients with symptomatic heart failure. Digoxin appears to be of most benefit in patients with severe heart failure, cardiomegaly and a third heart sound. Digoxin should be used as a second-line drug after diuretics, angiotensin-converting enzyme inhibitors and beta-blockers in patients with congestive heart failure who are in sinus rhythm. Digoxin should be used as a first-line drug in patients with congestive heart failure who are in atrial fibrillation. ARRHYTHMIAS: Digoxin has a limited, but useful, role, either alone or in combination with other agents such as beta-blockers, diltiazem or verapamil, in achieving satisfactory resting ventricular rate control in patients with chronic atrial fibrillation. In patients who lead a predominantly sedentary lifestyle (perhaps particularly in those who are elderly), digoxin alone may be the agent of choice.

Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1.

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.