RESUMEN
INTRODUCTION: The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative proposes a three-step approach to evaluate proarrhythmogenic liability of drug candidates: effects on individual ion channels in heterologous expression systems, integrating these data into in-silico models of the electrical activity of human cardiomyocytes, and comparison with experiments on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Here we introduce patch-clamp electrophysiology techniques on hiPSC-CM to combine two of the CiPA steps in one assay. METHODS: We performed automated patch-clamp experiments on hiPSC-CM (Cor.4U®, Ncardia) using the CytoPatch™2 platform in ruptured whole-cell and ß-escin-perforated-patch configurations. A combination of three voltage-clamp protocols allowed recording of five distinct ion current components (voltage-gated Na+ current, L-type Ca2+ current, transient outward K+ current, delayed rectifier K+ current, and "funny" hyperpolarization-activated current) from the same cell. We proved their molecular identity by either Na+ replacement with choline or by applying specific blockers: nifedipine, cisapride, chromanol 293B, phrixotoxin-1, ZD7288. We developed a C++ script for automated analysis of voltage-clamp recordings and computation of ion current/conductance surface density for these five cardiac ion currents. RESULTS: The distributions from nâ¯=â¯54 hiPSC-CM in "ruptured" patch-clamp vs. nâ¯=â¯35 hiPSC-CM in ß-escin-perforated patch-clamp were similar for membrane capacitance, access resistance, and ion current/conductance surface densities. The ß-escin-perforated configuration resulted in improved stability of action potential (AP) shape and duration over a 10-min interval, with APD90 decay rate 0.7⯱â¯1.6%/min (mean⯱â¯SD, nâ¯=â¯4) vs. 4.6⯱â¯1.1%/min. (nâ¯=â¯3) for "ruptured" approach (pâ¯=â¯0.0286, one-tailed Mann-Whitney test). DISCUSSION: The improved stability obtained here will allow development of CiPA-compliant automated patch-clamp assays on hiPSC-CM. Future applications include the study of multi ion-channel blocking properties of drugs using dynamic-clamp protocols, adding a valuable new tool to the arsenal of safety-pharmacology.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Arritmias Cardíacas/inducido químicamente , Canales Iónicos/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Arritmias Cardíacas/diagnóstico , Simulación por Computador , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Humanos , Células Madre Pluripotentes Inducidas/citología , Canales Iónicos/metabolismo , Miocitos Cardíacos/citología , Técnicas de Placa-ClampRESUMEN
While mutations in the SNCA gene (α-synuclein [α-syn]) are causal in rare familial forms of Parkinson's disease (PD), the prevalence of α-syn aggregates in the cortices of sporadic disease cases emphasizes the need to understand the link between α-syn accumulation and disease pathogenesis. By employing a combination of human pluripotent stem cells (hPSCs) that harbor the SNCA-A53T mutation contrasted against isogenic controls, we evaluated the consequences of α-syn accumulation in human A9-type dopaminergic (DA) neurons (hNs). We show that the early accumulation of α-syn in SNCA-A53T hNs results in changes in gene expression consistent with the expression profile of the substantia nigra (SN) from PD patients, analyzed post mortem. Differentially expressed genes from both PD patient SN and SNCA-A53T hNs were associated with regulatory motifs transcriptionally activated by the antioxidant response pathway, particularly Nrf2 gene targets. Differentially expressed gene targets were also enriched for gene ontologies related to microtubule binding processes. We thus assessed the relationship between Nrf2-mediated gene expression and neuritic pathology in SNCA-A53T hNs. We show that SNCA-mutant hNs have deficits in neuritic length and complexity relative to isogenic controls as well as contorted axons with Tau-positive varicosities. Furthermore, we show that mutant α-syn fails to complex with protein kinase C (PKC), which, in turn, results in impaired activation of Nrf2. These neuritic defects result from impaired Nrf2 activity on antioxidant response elements (AREs) localized to a microtubule-associated protein (Map1b) gene enhancer and are rescued by forced expression of Map1b as well as by both Nrf2 overexpression and pharmaceutical activation in PD neurons.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Factor 2 Relacionado con NF-E2/genética , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Animales , Elementos de Respuesta Antioxidante/genética , Axones/efectos de los fármacos , Axones/patología , Diferenciación Celular/genética , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Elementos de Facilitación Genéticos , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Mutación , Neuritas/metabolismo , Neuritas/patología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Proteína Quinasa C/genética , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
OBJECTIVE: Antipsychotic drugs target dopamine and serotonin receptors as well as Kv11.1 potassium channels encoded by KCNH2. Variable patient responses and a wide range of side effects, however, limit their efficacy. Slow metabolizer status and gene variants in KCNH2 associated with increased expression of Kv11.1-3.1, an alternatively spliced isoform of Kv11.1, are correlated with improved responses to antipsychotic medications. Here, the authors test the hypothesis that these effects may be influenced by differential drug binding to Kv11.1 channel isoforms. METHOD: Drug block of Kv11.1 isoforms was tested in cellular electrophysiology assays. The effects of drug metabolism and KCNH2 genotypes on clinical responses were assessed in patients enrolled in the multicenter Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE). RESULTS: Risperidone caused greater in vitro block of the alternatively spliced Kv11.1-3.1 isoform than full-length Kv11.1-1A channels, whereas its metabolite paliperidone and other atypical antipsychotics have similar potencies for the two isoforms. In the CATIE study (N=362), patients with genotypes associated with increased Kv11.1-3.1 expression (N=52) showed a better treatment response to risperidone compared with other drugs, but this association was dependent on metabolism status. Patients with KCNH2 risk genotypes and slow metabolizer status (approximately 7% of patients) showed marked improvement in symptoms when treated with risperidone compared with patients with fast metabolizer status or without the KCNH2 risk genotypes. CONCLUSIONS: These data support the hypothesis that Kv11.1 channels play a role in the therapeutic action of antipsychotic drugs, particularly risperidone, and further highlight the promise of optimizing response with genotype-guided therapy for schizophrenia patients.
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Canales de Potasio Éter-A-Go-Go/genética , Risperidona , Esquizofrenia , Adulto , Empalme Alternativo , Antipsicóticos/metabolismo , Antipsicóticos/farmacocinética , Canal de Potasio ERG1 , Femenino , Genotipo , Humanos , Masculino , Isoformas de Proteínas , Risperidona/metabolismo , Risperidona/farmacocinética , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Esquizofrenia/metabolismo , Resultado del TratamientoRESUMEN
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.
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Clozapina/farmacocinética , Canales de Potasio Éter-A-Go-Go/agonistas , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacocinética , Animales , Células CHO , Células Cultivadas , Clozapina/metabolismo , Cricetinae , Cricetulus , Interacciones Farmacológicas/fisiología , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Bloqueadores de los Canales de Potasio/metabolismoRESUMEN
Loss of Kv11.1 potassium channel function is the underlying cause of pathology in long-QT syndrome type 2, one of the commonest causes of sudden cardiac death in the young. Previous studies have identified the cytosolic PAS (Per/Arnt/Sim) domain as a hotspot for mutations that cause Kv11.1 trafficking defects. To investigate the underlying basis of this observation, we have quantified the effect of mutants on domain folding as well as interactions between the PAS domain and the remainder of the channel. Apart from R56Q, all mutants impaired the thermostability of the isolated PAS domain. Six mutants, located in the vicinity of a hydrophobic patch on the PAS domain surface, also affected binding of the isolated PAS domain to an N-terminal truncated hERG (human ether-a-go-go-related gene) channel. Conversely, four other surface mutants (C64Y, T65P, A78P and I96T) and one buried mutant (L86R) did not prevent the isolated PAS domain binding to the truncated channels. Our results highlight a critical role for interactions between the PAS domain and the remainder of the channel in the hERG assembly and that mutants that affect PAS domain interactions with the remainder of the channel have a more severe trafficking defect than that caused by domain unfolding alone.
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Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Mutación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/química , Femenino , Células HEK293 , Humanos , Unión Proteica/genética , Proteínas Serina-Treonina Quinasas/química , Transporte de Proteínas/genética , Xenopus laevisRESUMEN
Recent genome wide association studies identified a brain and primate specific isoform of a voltage-gated potassium channel, referred to as Kv11.1-3.1, which is significantly associated with schizophrenia. The 3.1 isoform replaces the first 102 amino acids of the most abundant isoform (referred to as Kv11.1-1A) with six unique amino acids. Here we show that the Kv11.1-3.1 isoform has faster rates of channel deactivation but a slowing of the rates of inactivation compared to the Kv11.1-1A isoform. The Kv11.1-3.1 isoform also has a significant depolarizing shift in the voltage-dependence of steady-state inactivation. The consequence of the altered gating kinetics is that there is lower current accumulation for Kv11.1-3.1 expressing cells during repetitive action potential firing compared to Kv11.1-1A expressing cells, which in turn will result in longer lasting trains of action potentials. Increased expression of Kv11.1-3.1 channels in the brain of schizophrenia patients might therefore contribute to disorganized neuronal firing.