Interaction of the S6 proline hinge with N-type and C-type inactivation in Kv1.4 channels.
Interaction of the S6 proline hinge with N-type and C-type inactivation in Kv1.4 channels.
复制标题
S6 脯氨酸铰链与 Kv1.4 通道中 N 型和 C 型失活的相互作用。
DOI：
10.1016/j.bpj.2012.08.036
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发表时间：
2012
影响因子：
3.4
通讯作者：
Rasmusson,RandallL
中科院分区：
文献类型：
--
作者：
Bett,GlennaCL;Lis,Agnieszka;Guo,Hong;Liu,MiMi;Zhou,Qinlian;Rasmusson,RandallL
几个电压门控通道在 S6 的细胞内侧共享一个脯氨酸-缬氨酸-脯氨酸（脯氨酸铰链）序列基序。我们研究了 Kv1.4 通道中的脯氨酸铰链，它通过两种机制失活：N 型和 C 型。我们将第二个脯氨酸突变为甘氨酸或丙氨酸：P558A、P558G。在 N 末端存在/不存在的情况下研究这些突变，以分离脯氨酸铰链与 N 型和 C 型失活之间相互作用的影响。两种 S6 突变均减缓或消除了 N 型和 C 型失活，并改变了失活的恢复。 P558G 将激活以及 N 型和 C 型失活减慢了近一个数量级。对细胞外酸中毒和细胞内奎尼丁结合的敏感性仍然存在，表明 N 型和 C 型失活中的跨膜通讯得以保留，这与我们之前在 C 型失活期间涉及 S6 的主要结构重排的发现一致。 P558A 具有很强的破坏性：激活速度减慢了一个数量级以上，并且没有观察到失活。这些结果与我们的假设一致，即脯氨酸铰链和细胞内 S6 运动在失活和恢复中发挥重要作用。计算机模型表明，P558G 和 P558A 突变都会修改早期的电压依赖性步骤，并做出最终的电压不敏感步骤，该步骤限制正电位的速率。
Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.