Tseng G N, Jiang M, Yao J A
Department of Pharmacology, Columbia University, New York, New York, USA.
J Pharmacol Exp Ther. 1996 Nov;279(2):865-76.
4-Aminopyridine (4AP) can block various K channels with different state dependences; block occurs in the activated state or in the closed state. The use of K channel clones to study the mechanism and structural determinants responsible for the state dependence of 4AP actions has been hampered by the fact that, for all the K channel clones examined so far, 4AP binding and unbinding occur mainly in the activated state. We report here that 4AP binding to a fast inactivating K channel encoded by Kv4.2 in Xenopus oocytes occurred exclusively in the closed state. The binding rate was slow and independent of membrane voltage in the range from -80 to -120 mV. The binding rate was linearly related to 4AP concentration, yielding apparent binding and unbinding rate constants of 0.012 mM-1 s-1 and 0.062 s-1, respectively. 4AP dissociation from Kv4.2 occurred in two processes, a slow process in the closed state (in a voltage range from -70 to -40 mV) and a fast process in the activated state, which suggested that the closure of the activation gate of Kv4.2 did not prevent the entry or exit of 4AP molecules but slowed these processes. 4AP slowed the rate of Kv4.2 decay during depolarization, consistent with the notion that channel inactivation occurred only after 4AP dissociation. Inactivating Kv4.2 channels prevented 4AP binding. Therefore, 4AP binding and Kv4.2 inactivation were mutually exclusive. This, in conjunction with the observation that 4AP blocked Kv4.2 channels from the intracellular side of the cell membrane, suggests that the 4AP binding site is on the cytoplasmic surface of the Kv4.2 channel at, or adjacent to, the domains involved in channel inactivation. The distinct features of 4AP actions on the time course of transient outward current in human ventricular myocytes suggest that Kv4.2-like subunits are important in the formation of these channels in human heart.
4-氨基吡啶（4AP）能够阻断各种具有不同状态依赖性的钾通道；阻断作用发生在激活状态或关闭状态。利用钾通道克隆来研究4AP作用的状态依赖性的机制和结构决定因素，一直受到这样一个事实的阻碍，即对于迄今为止所检测的所有钾通道克隆，4AP的结合和解离主要发生在激活状态。我们在此报告，4AP与非洲爪蟾卵母细胞中由Kv4.2编码的快速失活钾通道的结合仅发生在关闭状态。结合速率缓慢，且在-80至-120 mV的膜电压范围内与膜电压无关。结合速率与4AP浓度呈线性关系，表观结合和解离速率常数分别为0.012 mM-1 s-1和0.062 s-1。4AP从Kv4.2的解离发生在两个过程中，一个是关闭状态下的缓慢过程（在-70至-40 mV的电压范围内），另一个是激活状态下的快速过程，这表明Kv4.2激活门的关闭并未阻止4AP分子的进入或离开，只是减缓了这些过程。4AP减缓了去极化过程中Kv4.2的衰减速率，这与通道失活仅在4AP解离后发生的观点一致。失活的Kv4.2通道可阻止4AP结合。因此，4AP结合和Kv4.2失活是相互排斥的。这一点，再加上4AP从细胞膜内侧阻断Kv4.2通道的观察结果，表明4AP结合位点位于Kv4.2通道细胞质表面，在参与通道失活的结构域处或其附近。4AP对人心室肌细胞瞬时外向电流时间进程的作用的独特特征表明，Kv4.2样亚基在人心脏中这些通道的形成中很重要。
