Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain
Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain
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DOI：
10.1038/sj.bjp.0702477
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发表时间：
1999-04-01
影响因子：
7.3
通讯作者：
Franciolini, F
中科院分区：
文献类型：
--
作者：
Catacuzzeno, L;Trequattrini, C;Franciolini, F
1 使用全细胞膜片钳配置研究维拉帕米阻断鸡背根神经节 (DRG) 神经元延迟整流 K 电流 (I-K(DR)) 的机制。我们特别关注阻断位点的位置，并且使用永久带电维拉帕米类似物 D890 的维拉帕米活性形式（中性或带电）。D890 的阻断显示出与维拉帕米相似的特征，表明阻断具有相同的状态依赖性性质。与维拉帕米相比，D890 仅在内部应用时才有效，并且其阻断是电压依赖性的（开启速率的 136 mV/e 倍变化）。鉴于维拉帕米阻滞对电压不敏感（Trequattrini 等，1998），这些观察结果表明维拉帕米以不带电形式到达其结合位点，并从细胞质进入结合域。3 在外部 K 和饱和维拉帕米中，我们记录的尾电流不会单调衰减，而是显示出初始增加（钩）。由于只有当维拉帕米解阻显着依赖于电压时才能观察到这些电流，因此有人建议（DeCoursey，1995）中性药物在结合后被质子化。我们通过评估维拉帕米和 D890 的钩状尾电流解阻断率的电压依赖性来测试这一假设。4 通过采用 Na 通道离子阻断的经典 Woodhull (1973) 模型，很好地描述了 D890（而非维拉帕米）解阻断率的电压依赖性。维拉帕米解离速率的电压依赖性与动力学方案一致，其中结合的药物可以通过快速平衡质子化？带电和中性的维拉帕米都可以从该位点解开，但具有不同的动力学和电压依赖性。
1 The mechanism of verapamil block of the delayed rectifier K currents (I-K(DR)) in chick dorsal root ganglion (DRG) neurons was investigated using the whole-cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890.2 Block by D890 displayed similar characteristics to that of verapamil, indicating the same state-dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e-fold change of the on rate). Given that verapamil block is insensitive to voltage (Trequattrini et al., 1998), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm.3 In external K and saturating verapamil we recorded tail currents that did not decay monotonicalIy but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested (DeCoursey, 1995) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890.4 The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium? and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies.