Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Biol Chem. 2013 May 10;288(19):13741-7. doi: 10.1074/jbc.M113.462366. Epub 2013 Mar 27.
In-frame deletion mutation (Del-L955) in NaV1.7 sodium channel from a kindred with erythromelalgia hyperpolarizes activation.
Del-L955 twists the S6 helix, displacing the Phe960 activation gate. Replacement of Phe960 at the correct helical position depolarizes activation.
Radial tuning of the activation gate is critical to the activation of NaV1.7 channel.
Structural modeling guided electrophysiology reveals the functional importance of radial tuning of the S6 segment. Voltage-gated sodium (NaV) channels are membrane proteins that consist of 24 transmembrane segments organized into four homologous domains and are essential for action potential generation and propagation. Although the S6 helices of NaV channels line the ion-conducting pore and participate in channel activation, their functional architecture is incompletely understood. Our recent studies show that a naturally occurring in-frame deletion mutation (Del-L955) of NaV1.7 channel, identified in individuals with a severe inherited pain syndrome (inherited erythromelalgia) causes a substantial hyperpolarizing shift of channel activation. Here we took advantage of this deletion mutation to understand the role of the S6 helix in the channel activation. Based on the recently published structure of a bacterial NaV channel (NaVAb), we modeled the WT and Del-L955 channel. Our structural model showed that Del-L955 twists the DII/S6 helix, shifting location and radial orientation of the activation gate residue (Phe(960)). Hypothesizing that these structural changes produce the shift of channel activation of Del-L955 channels, we restored a phenylalanine in wild-type orientation by mutating Ser(961) (Del-L955/S961F), correcting activation by ∼10 mV. Correction of the displaced Phe(960) (F960S) together with introduction of the rescuing activation gate residue (S961F) produced an additional ∼6-mV restoration of activation of the mutant channel. A simple point mutation in the absence of a twist (L955A) did not produce a radial shift and did not hyperpolarize activation. Our results demonstrate the functional importance of radial tuning of the sodium channel S6 helix for the channel activation.
红斑性肢痛症相关的钠离子通道 NaV1.7 中的框内缺失突变（Del-L955）使激活超极化。
Del-L955 扭曲 S6 螺旋，使 Phe960 激活门发生位移。在正确的螺旋位置替换 Phe960 使激活去极化。
激活门的径向调谐对 NaV1.7 通道的激活至关重要。
结构建模指导的电生理学揭示了 S6 片段的径向调谐的功能重要性。电压门控钠离子（NaV）通道是由 24 个跨膜片段组成的膜蛋白，分为四个同源结构域，是动作电位产生和传播所必需的。尽管 NaV 通道的 S6 螺旋排列在离子传导孔中并参与通道激活，但它们的功能结构仍不完全清楚。我们最近的研究表明，在患有严重遗传性疼痛综合征（遗传性红斑性肢痛症）的个体中发现的 NaV1.7 通道的一种天然存在的框内缺失突变（Del-L955）导致通道激活发生显著的超极化移位。在这里，我们利用这种缺失突变来了解 S6 螺旋在通道激活中的作用。基于最近发表的细菌 NaV 通道（NaVAb）结构，我们对 WT 和 Del-L955 通道进行建模。我们的结构模型表明，Del-L955 扭曲了 DII/S6 螺旋，改变了激活门残基（Phe（960））的位置和径向方向。假设这些结构变化导致 Del-L955 通道的通道激活移位，我们通过突变 Ser（961）（Del-L955/S961F）以野生型取向恢复一个苯丙氨酸，将激活纠正约 10 mV。纠正移位的 Phe（960）（F960S）并引入修复激活门残基（S961F）使突变通道的激活进一步恢复约 6 mV。在不存在扭曲的情况下进行简单的点突变（L955A）不会产生径向移位，也不会使激活超极化。我们的结果表明，钠离子通道 S6 螺旋的径向调谐对通道激活具有重要的功能意义。
