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Block of the lymphocyte K(+) channel mKv1.3 by the phenylalkylamine verapamil: kinetic aspects of block and disruption of accumulation of block by a single point mutation.

ABSTRACT: 1. Phenylalkylamines (PAA) usually known for their action on L-type Ca(2+) channels potently block the C-type inactivating lymphocyte Kv1.3 channel resulting in inhibition of activation of T lymphocytes. In order to design PAAs blocking Kv1.3 specifically over L-type Ca(2+) channels, we investigated the state-dependent manner of mKv1. 3 block by the PAA verapamil. 2. Verapamil seems to have access to the open state (OB) and, once bound to the channel, the channel-verapamil complex is absorbed into a slowly recovering state. This state was proposed to be the inactivated blocked state (IB). Here we present a quantitative description of the transition into this state and provide evidence for the IB state through experiments with an inactivation lacking mutant channel. Since the inactivated state cannot be reached in this case the IB state cannot be reached either. 3. We show that the transition OB-->IB is accelerated by verapamil most likely through a mechanism involving the reduction of [K(+)] at an inactivation modulating low affinity binding site for K(+) at the outer vestibule. 4. Measurements of the voltage-dependence of the off-rate constants for verapamil suggest that verapamil can reach the channel in its neutral form and might get partially protonated while bound. Thus only those verapamil molecules that are protonated can more easily dissociate at hyperpolarizing voltages. 5. Since open block kinetics were shown to be similar for wild type mKv1.3 and the H404T mutant mKv1.3 channel, and since the block of the H404T mutant channels by verapamil could be described exactly by a simple three-state open block model, the mutant channel could serve as a screening channel to determine open block affinities of new PAA derivatives in high through-put experiments.

SUBMITTER: Robe RJ

PROVIDER: S-EPMC1572478 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Project description:Accurate diagnosis of predisposition to long QT syndrome is crucial for reducing the risk of cardiac arrhythmias. In recent years, drug-induced provocative tests have proved useful to unmask some latent mutations linked to cardiac arrhythmias. In this study we expanded this concept by developing a prototype for a computational provocative screening test to reveal genetic predisposition to acquired long-QT syndrome (aLQTS). We developed a computational approach to reveal the pharmacological properties of I(Kr) blocking drugs that are most likely to cause aLQTS in the setting of subtle alterations in I(Kr) channel gating that would be expected to result from benign genetic variants.Weused themodel to predict themost potentially lethal combinations of kinetic anomalies and drug properties. In doing so, we also implicitly predicted ideal inverse therapeutic properties of K channel openers that would be expected to remedy a specific defect. We systematically performed “in silico mutagenesis” by altering discrete kinetic transition rates of the Fink et al. Markov model of human I(Kr) channels, corresponding to activation, inactivation, deactivation and recovery from inactivation of I(Kr) channels. We then screened and identified the properties of I(Kr) blockers that caused acquired long QT and therefore unmasked mutant phenotypes formild,moderate and severe variants. Mutant I(Kr) channels were incorporated into the O'Hara et al. human ventricular action potential (AP) model and subjected to simulated application of a wide variety of I(Kr)-drug interactions in order to identify the characteristics that selectively exacerbate the AP duration (APD) differences between wild-type and I(Kr) mutated cells. Our results show that drugs with disparate affinities to conformation states of the I(Kr) channel are key to amplify variants underlying susceptibility to acquired long QT syndrome, an effect that is especially pronounced at slow frequencies. Finally, we developed a mathematical formulation of the M54T MiRP1 latent mutation and simulated a provocative test. In this setting, application of dofetilide dramatically amplified the predicted QT interval duration in the M54T hMiRP1 mutation compared to wild-type.

Project description:Accurate diagnosis of predisposition to long QT syndrome is crucial for reducing the risk of cardiac arrhythmias. In recent years, drug-induced provocative tests have proved useful to unmask some latent mutations linked to cardiac arrhythmias. In this study we expanded this concept by developing a prototype for a computational provocative screening test to reveal genetic predisposition to acquired long-QT syndrome (aLQTS). We developed a computational approach to reveal the pharmacological properties of IKr blocking drugs that are most likely to cause aLQTS in the setting of subtle alterations in IKr channel gating that would be expected to result from benign genetic variants. We used the model to predict the most potentially lethal combinations of kinetic anomalies and drug properties. In doing so, we also implicitly predicted ideal inverse therapeutic properties of K channel openers that would be expected to remedy a specific defect. We systematically performed "in silico mutagenesis" by altering discrete kinetic transition rates of the Fink et al. Markov model of human IKr channels, corresponding to activation, inactivation, deactivation and recovery from inactivation of IKr channels. We then screened and identified the properties of IKr blockers that caused acquired long QT and therefore unmasked mutant phenotypes for mild, moderate and severe variants. Mutant IKr channels were incorporated into the O'Hara et al. human ventricular action potential (AP) model and subjected to simulated application of a wide variety of IKr-drug interactions in order to identify the characteristics that selectively exacerbate the AP duration (APD) differences between wild-type and IKr mutated cells. Our results show that drugs with disparate affinities to conformation states of the IKr channel are key to amplify variants underlying susceptibility to acquired long QT syndrome, an effect that is especially pronounced at slow frequencies. Finally, we developed a mathematical formulation of the M54T MiRP1 latent mutation and simulated a provocative test. In this setting, application of dofetilide dramatically amplified the predicted QT interval duration in the M54T hMiRP1 mutation compared to wild-type.

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Block of the lymphocyte K(+) channel mKv1.3 by the phenylalkylamine verapamil: kinetic aspects of block and disruption of accumulation of block by a single point mutation.

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