Stabilizing the closed S6 gate in the Shaker Kv channel through modification of a hydrophobic seal.
Stabilizing the closed S6 gate in the Shaker Kv channel through modification of a hydrophobic seal.
复制标题
通过修改疏水密封，稳定振动筛KV通道中的闭合S6门。
DOI：
10.1085/jgp.200409098
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发表时间：
2004-10
影响因子：
3.8
通讯作者：
Swartz, Kenton J
中科院分区：
文献类型：
--
作者：
Kitaguchi, Tetsuya;Sukhareva, Manana;Swartz, Kenton J
K+通道的主要激活门被认为位于离子传导孔的细胞内入口附近。在之前对Shaker中S6激活门的研究中，我们发现V478到W的突变导致了一个不能传导离子的通道，尽管电压传感器能够响应膜去极化而转移门控电荷。在本研究中，我们探索了V478W中不导电表型的机制，并将其与W434F进行了比较，W434F是一种位于孔的细胞外区域的突变，由于通道主要处于失活状态，该区域不导电。我们首先使用与细胞内孔相互作用的探针检查细胞内门是否移动，并通过研究能够传导离子的异二聚体通道的失活特性。这些实验结果支持了W434F和V478W中不同的不传导机制，表明V478W中的栅极要么保持关闭状态，要么突变在打开状态下对离子渗透产生了很大的屏障。异二聚体和离子传导恢复的双突变构建体的单通道记录表明，V478W突变不会显著改变单一电导。综上所述，我们的研究结果表明，V478W突变导致了从封闭到开放平衡向封闭状态的深刻转变。在K+通道中这一关键区域的结构背景下讨论了这一机制。
The primary activation gate in K+ channels is thought to reside near the intracellular entrance to the ion conduction pore. In a previous study of the S6 activation gate in Shaker, we found that mutation of V478 to W results in a channel that cannot conduct ions even though the voltage sensors are competent to translocate gating charge in response to membrane depolarization. In the present study we explore the mechanism underlying the nonconducting phenotype in V478W and compare it to that of W434F, a mutation located in an extracellular region of the pore that is nonconducting because the channel is predominantly found in an inactivated state. We began by examining whether the intracellular gate moves using probes that interact with the intracellular pore and by studying the inactivation properties of heterodimeric channels that are competent to conduct ions. The results of these experiments support distinct mechanisms underlying nonconduction in W434F and V478W, suggesting that the gate in V478W either remains closed, or that the mutation has created a large barrier to ion permeation in the open state. Single channel recordings for heterodimeric and double mutant constructs in which ion conduction is rescued suggest that the V478W mutation does not dramatically alter unitary conductance. Taken together, our results suggest that the V478W mutation causes a profound shift of the closed to open equilibrium toward the closed state. This mechanism is discussed in the context of the structure of this critical region in K+ channels.