Piper David R, Hinz William A, Tallurri Chandra K, Sanguinetti Michael C, Tristani-Firouzi Martin
Department of Physiology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah 84112, USA.
J Biol Chem. 2005 Feb 25;280(8):7206-17. doi: 10.1074/jbc.M411042200. Epub 2004 Nov 4.
Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly(514)-Tyr(545)) that comprise the S4 domain and the flanking S3-S4 and S4-S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as |DeltaDeltaG| > 1.0 kcal/mol) on a three-dimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4-S5 linkers and the C-terminal end of S4 perturbed activation (|DeltaDeltaG| > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating.
缓慢激活和快速C型失活导致人类醚 - 去极化相关基因（hERG）通道的电流 - 电压关系出现内向整流。为了表征与hERG激活和失活相关的电压传感器运动，我们对构成S4结构域以及侧翼S3 - S4和S4 - S5连接子的32个氨基酸（Gly(514)-Tyr(545)）进行了丙氨酸扫描。使用切开卵母细胞凡士林间隙和双微电极电压钳技术测量野生型和突变型通道的门控电流和离子电流，以确定电荷移动、激活和失活的电压依赖性。将电荷扰动突变（定义为|ΔΔG|>1.0 kcal/mol）的位置映射到三维S4同源模型上，揭示了一种螺旋模式。正如预期的那样，这些残基的突变也改变了激活。然而，S3 - S4和S4 - S5连接子以及S4 C末端的残基突变在不改变电荷移动的情况下扰动了激活（|ΔΔG|>1.0 kcal/mol），这表明这些区域的天然残基将S4运动与激活门的打开相耦合，或稳定通道的开放或关闭状态。最后，一组不同的残基突变影响失活，并映射到S4螺旋的一个没有激活扰动残基的面上。这些结果确定了S4电压传感器上对hERG激活和失活门控有不同贡献的区域。
