Reversed voltage-dependent gating of a bacterial sodium channel with proline substitutions in the S6 transmembrane segment
Reversed voltage-dependent gating of a bacterial sodium channel with proline substitutions in the S6 transmembrane segment
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DOI：
10.1073/pnas.0408270101
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发表时间：
2004-12-21
影响因子：
11.1
通讯作者：
Catterall, WA
中科院分区：
文献类型：
--
作者：
Zhao, Y;Scheuer, T;Catterall, WA
电压门控类离子通道超家族的成员具有保守的孔结构。排列在孔(M2或S6)上的跨膜螺旋被认为是通过在铰链甘氨酸残基上弯曲而在细胞质末端对其进行门控的。Pro残基有利于a-螺旋的弯曲，而用Pro取代该甘氨酸(G219)可显著稳定细菌Na+通道NaChBac的开放状态。在这里，我们用脯氨酸诱变的方法检测了NaChBac的S6孔衬残基。在15个脯氨酸替代突变体中，有5个产生了去极化激活的Na+通道，但只有G219P通道具有强烈的负移激活电压依赖关系，表明G219处的弯曲具有去极化激活门控的特异性。值得注意的是，与G219位于同一表面的S6的三个脯氨酸替代突变产生了在超极化时激活和非常缓慢地失活的通道。对L226P的研究表明，超极化至-147 mV时激活为一半，比WT多123 mV。组合突变分析和局麻药伊多卡因阻断研究支持超极化激活门控是由S6螺旋形成的细胞质门打开所致。用多种氨基酸替代L226表明，超极化激活的门控与高弯曲倾向相关，而去极化激活的门控有利于低弯曲倾向。我们的结果进一步确定了S6的弯曲不仅在决定电压依赖中起主导作用，而且在决定电压相关门的极性方面也起着主导作用。动物超极化和环核苷酸门控(HCN)通道和植物KAT通道的自然超极化激活门控可能涉及在类似的S6氨基酸残基上弯曲。
Members of the voltage-gated-like ion channel superfamily have a conserved pore structure. Transmembrane helices that line the pore (M2 or S6) are thought to gate it at the cytoplasmic end by bending at a hinge glycine residue. Proline residues favor bending of a-helices, and substitution of proline for this glycine (G219) dramatically stabilizes the open state of a bacterial Na+ channel NaChBac. Here we have probed S6 pore-lining residues of NaChBac by proline mutagenesis. Five of 15 proline-substitution mutants yielded depolarization-activated Na+ channels, but only G219P channels have strongly negatively shifted voltage dependence of activation, demonstrating specificity for bending at G219 for depolarization-activated gating. Remarkably, three proline-substitution mutations on the same face of S6 as G219 yielded channels that activated upon hyperpolarization and inactivated very slowly. Studies of L226P showed that hyperpolarization to - 147 mV gives half-maximal activation, 123 mV more negative than WT. Analysis of combination mutations and studies of block by the local anesthetic etidocaine favored the conclusion that hyperpolarization-activated gating results from opening of the cytoplasmic gate formed by S6 helices. Substitution of multiple amino acids for L226 indicated that hyperpolarization-activated gating was correlated with a high propensity for bending, whereas depolarization-activated gating was favored by a low propensity for bending. Our results further define the dominant role of bending of S6 in determining not only the voltage dependence but also the polarity of voltage-dependent gating. Native hyperpolarization-activated gating of hyperpolarization- and cyclic nucleotide-gated (HCN) channels in animals and KAT channels in plants may involve bending at analogous S6 amino acid residues.