Investigating the putative glycine hinge in Shaker potassium channel.
Investigating the putative glycine hinge in Shaker potassium channel.
复制标题
研究振荡器钾通道中推定的甘氨酸铰链。
DOI：
10.1085/jgp.200509287
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发表时间：
2005-09
影响因子：
3.8
通讯作者：
Horn, Richard
中科院分区：
文献类型：
--
作者：
Ding, Shinghua;Ingleby, Lindsey;Ahern, Christopher A;Horn, Richard
打开的钾通道的晶体结构揭示了排列在孔内螺旋的一个结（Jiang, yx, a . Lee， J.Y. Chen， M. Cadene， B.T. Chait和R. MacKinnon）。2002. 自然417:523 - 526)。假定的枢纽点是一个高度保守的甘氨酸残基。我们检测了同源残基（Gly466）在Shaker钾通道S6跨膜段中的作用。无功能的丙氨酸突变体G466A将与野生型（WT）亚基组装，尽管效果很差，但会抑制功能性表达。为了测试甘氨酸残基是否对激活门控至关重要，我们在G466A的背景下沿S6段进行了甘氨酸扫描。尽管所有这些双突变体都缺乏成熟的Shaker通道所特有的更高水平的糖基化，但其中一个（G466A/V467G）能够产生电压依赖性钾电流。表面生物素化表明含有G466A的功能性和非功能性构建体在质膜中的表达水平相当。与WT通道相比，移位的甘氨酸突变体在电压依赖性通道打开方面存在损伤，包括激活曲线右移和激活率下降。双突变体具有相对正常的开放通道特性，除了对细胞内阻滞剂的亲和力降低，这是Val467侧链缺失的结果。双突变体M440A/G466A和G466A/V467A的控制实验表明，Gly466提供的灵活性比其小尺寸更重要。我们的研究结果支持Gly466在通道的生物发生和激活门控中的铰链作用。
The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523–526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.