The mechanisms involved in the 4-aminopyridine (4-AP)-induced block of delayed rectifier K+ current (IK(V)) in vascular smooth muscle cells were studied in cells enzymatically isolated from the rabbit coronary artery. 2. 4-AP inhibited slowly inactivating IK(V) in a dose-dependent manner (concentration producing half-maximal inhibition, K1/2, = 1.37 mM), and shifted the steady-state activation and inactivation curves of IK(V) by +9 and +16 mV, respectively. 3. The time constant of activation was significantly increased by 4-AP at +20 mV; deactivation kinetics were unaffected upon repolarization to -40 mV. The fast (tau f approximately 1 s) and slow (tau s approximately 5 s) time constants of inactivation (0 and +20 mV), and the recovery kinetics (tau r approximately 6 s) at -60 mV were not significantly affected by 0.5 mM 4-AP. However, tau f disappeared in the presence of 2 mM 4-AP while tau s remained unaffected. 4. Use-dependent unblock of IK(V) was revealed at potentials > or = -10 mV from analyses of the voltage dependence of 4-AP-sensitive currents and the frequency-dependent changes ('reverse use dependence') of IK(V) during the application of repetitive steps (-60 to +20 mV for 250 ms at a rate of 0.25 Hz) in control conditions, in the presence of 0.5 mM 4-AP, and after washout of the drug. These results suggested that 4-AP preferentially binds to the channel in the closed state, and unbinding is promoted by transitions to the open state. 5. The channel was modelled as a simple three-state mathematical loop model incorporating single closed, open and inactivated states. The block by 4-AP was modelled as a state-dependent interaction with 4-AP primarily binding to the closed state. Computer simulations support the hypothesis that 4-AP-induced block of the delayed rectifier K+ (KV) channel in the closed state is relieved during membrane depolarization. 6. Closed state binding of 4-AP to the KV channel depolarizes vascular smooth muscle cells by shifting the activation curve of these channels to more positive potentials.
摘要
在从兔冠状动脉酶解分离的细胞中，研究了4-氨基吡啶（4-AP）诱导的血管平滑肌细胞延迟整流钾电流（IK(V)）阻断所涉及的机制。2. 4-AP以剂量依赖性方式缓慢抑制IK(V)（产生半数最大抑制的浓度，K1/2 = 1.37 mM），并分别使IK(V)的稳态激活和失活曲线正向移动9 mV和16 mV。3. 在+20 mV时，4-AP显著增加激活时间常数；复极化至-40 mV时，失活动力学不受影响。0.5 mM 4-AP对-60 mV时的快速（τf约1 s）和慢速（τs约5 s）失活时间常数（0和+20 mV）以及恢复动力学（τr约6 s）无显著影响。然而，在2 mM 4-AP存在时，τf消失而τs仍不受影响。4. 通过分析4-AP敏感电流的电压依赖性以及在对照条件下、0.5 mM 4-AP存在时和药物洗脱后重复步骤（-60至+20 mV，持续250 ms，频率0.25 Hz）期间IK(V)的频率依赖性变化（“反向使用依赖性”），发现在电位≥ -10 mV时IK(V)存在使用依赖性解阻断。这些结果表明，4-AP优先结合处于关闭状态的通道，向开放状态的转变促进了解离。5. 将该通道建模为一个简单的三态数学循环模型，包含单个关闭、开放和失活状态。4-AP的阻断被建模为一种状态依赖性相互作用，4-AP主要结合到关闭状态。计算机模拟支持这样的假设，即4-AP诱导的延迟整流钾（KV）通道在关闭状态下的阻断在膜去极化过程中得到缓解。6. 4-AP与KV通道在关闭状态下的结合通过将这些通道的激活曲线向更正电位移动，使血管平滑肌细胞去极化。
