Zhu Yujie, Kyle John W, Lee Peter J
Department of Medicine and Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL 60612, USA.
Am J Physiol Heart Circ Physiol. 2006 Jul;291(1):H29-37. doi: 10.1152/ajpheart.01317.2005. Epub 2006 Feb 24.
A long QT mutation in the cardiac sodium channel, D1790G (DG), shows enhanced flecainide use-dependent block (UDB). The relative importance of open and inactivated states of the channel in flecainide UDB has been controversial. We used a modifiable, inactivation-deficient mutant channel that contains the F1486C mutation in the IFM motif to investigate the UDB difference between the wild-type (WT-ICM) and DG (DG-ICM) channels. UDB at 5 Hz was greater in DG-ICM than WT-ICM, and IC50 values for steady-state UDB were 7.19 and 18.06 microM, respectively. When [2-(trimethyammonium) ethyl]methanethiosulfonate bromide (MTSET) was included in the pipette and fast inactivation was disabled, IC50 was 5.04 microM for DG-ICM and 12.63 microM for WT-ICM. We measured open-channel block by flecainide directly in MTSET-treated, noninactivating ICM channels. Steady-state block was higher for DG-ICM than WT-ICM (IC50 was 2.34 microM for DG-ICM and 5.87 microM for WT-ICM), suggesting that open-channel block is an important determinant of flecainide UDB. We obtained association (kon) and dissociation (koff) rates for open-channel block by the Langmuir-isotherm model. They were koff = 31.37 s(-1), kon = 5.83 s(-1).microM(-1), and calculated Kd = 5.38 microM for WT-ICM (where Kd = koff/kon); and koff = 24.88 s(-1), kon = 9.54 s(-1).microM(-1), and calculated Kd = 2.61 microM for DG-ICM. These Kd values were similar to IC50 measured from steady-state open-channel block. Furthermore, we modeled UDB mathematically by using these kinetic rates and found that the model predicted experimental UDB accurately. The recovery from UDB had a minor contribution to UDB. Flecainide UDB is predominantly determined by an open-channel blocking mechanism, and DG-ICM channels appeared to have an altered open-channel state with higher flecainide affinity than WT-ICM.
心脏钠通道中的一种长QT突变，D1790G（DG），表现出增强的氟卡尼使用依赖性阻滞（UDB）。通道的开放状态和失活状态在氟卡尼UDB中的相对重要性一直存在争议。我们使用了一种可修饰的、缺乏失活功能的突变通道，该通道在IFM基序中含有F1486C突变，以研究野生型（WT-ICM）和DG（DG-ICM）通道之间的UDB差异。在5Hz时，DG-ICM的UDB大于WT-ICM，稳态UDB的IC50值分别为7.19和18.06 microM。当移液管中加入[2-（三甲基铵）乙基]甲硫代磺酸溴（MTSET）且快速失活被禁用时，DG-ICM的IC50为5.04 microM，WT-ICM的IC50为12.63 microM。我们在MTSET处理的、非失活的ICM通道中直接测量了氟卡尼对开放通道的阻滞。DG-ICM的稳态阻滞高于WT-ICM（DG-ICM的IC50为2.34 microM，WT-ICM的IC50为5.87 microM），这表明开放通道阻滞是氟卡尼UDB的一个重要决定因素。我们通过Langmuir等温线模型获得了开放通道阻滞的结合（kon）和解离（koff）速率。对于WT-ICM，它们分别为koff = 31.37 s(-1)，kon = 5.83 s(-1).microM(-1)，计算得出的Kd = 5.38 microM（其中Kd = koff/kon）；对于DG-ICM，koff = 24.88 s(-1)，kon = 9.54 s(-1).microM(-1)，计算得出的Kd = 2.61 microM。这些Kd值与从稳态开放通道阻滞测量得到的IC50相似。此外，我们使用这些动力学速率对UDB进行了数学建模，发现该模型能够准确预测实验性UDB。UDB的恢复对UDB的贡献较小。氟卡尼UDB主要由开放通道阻滞机制决定，并且DG-ICM通道似乎具有改变的开放通道状态，对氟卡尼的亲和力高于WT-ICM。
