Zhang Mei, Liu Jie, Tseng Gea-Ny
Department of Physiology, Virginia Commonwealth University, 1101 E. Marshall St., Richmond, VA 23298, USA.
J Gen Physiol. 2004 Dec;124(6):703-18. doi: 10.1085/jgp.200409119. Epub 2004 Nov 15.
The hERG channel has a relatively slow activation process but an extremely fast and voltage-sensitive inactivation process. Direct measurement of hERG's gating current (Piper, D.R., A. Varghese, M.C. Sanguinetti, and M. Tristani-Firouzi. 2003. PNAS. 100:10534-10539) reveals two kinetic components of gating charge transfer that may originate from two channel domains. This study is designed to address three questions: (1) which of the six positive charges in hERG's major voltage sensor, S4, are responsible for gating charge transfer during activation, (2) whether a negative charge in the cytoplasmic half of S2 (D466) also contributes to gating charge transfer, and (3) whether S4 serves as the sole voltage sensor for hERG inactivation. We individually mutate S4's positive charges and D466 to cysteine, and examine (a) effects of mutations on the number of equivalent gating charges transferred during activation (z(a)) and inactivation (z(i)), and (b) sidedness and state dependence of accessibility of introduced cysteine side chains to a membrane-impermeable thiol-modifying reagent (MTSET). Neutralizing the outer three positive charges in S4 and D466 in S2 reduces z(a), and cysteine side chains introduced into these positions experience state-dependent changes in MTSET accessibility. On the other hand, neutralizing the inner three positive charges in S4 does not affect z(a). None of the charge mutations affect z(i). We propose that the scheme of gating charge transfer during hERG's activation process is similar to that described for the Shaker channel, although hERG has less gating charge in its S4 than in Shaker. Furthermore, channel domain other than S4 contributes to gating charge involved in hERG's inactivation process.
hERG通道具有相对缓慢的激活过程，但具有极快且电压敏感的失活过程。对hERG门控电流的直接测量（Piper, D.R., A. Varghese, M.C. Sanguinetti, and M. Tristani-Firouzi. 2003. 《美国国家科学院院刊》. 100:10534 - 10539）揭示了门控电荷转移的两个动力学成分，这可能源于两个通道结构域。本研究旨在解决三个问题：（1）hERG主要电压感受器S4中的六个正电荷中，哪些在激活过程中负责门控电荷转移；（2）S2胞质侧的一个负电荷（D466）是否也对门控电荷转移有贡献；（3）S4是否作为hERG失活的唯一电压感受器。我们将S4的正电荷和D466分别突变为半胱氨酸，并研究（a）突变对激活过程中转移的等效门控电荷数（z(a)）和失活过程中转移的等效门控电荷数（z(i)）的影响，以及（b）引入的半胱氨酸侧链对膜不透性硫醇修饰试剂（MTSET）的可及性的方向性和状态依赖性。中和S4外侧的三个正电荷以及S2中的D466会降低z(a)，并且引入这些位置的半胱氨酸侧链在MTSET可及性方面经历状态依赖性变化。另一方面，中和S4内侧的三个正电荷不会影响z(a)。没有一个电荷突变影响z(i)。我们提出，尽管hERG的S4中的门控电荷比Shaker通道少，但hERG激活过程中的门控电荷转移模式与Shaker通道中描述的相似。此外，除S4之外的通道结构域对hERG失活过程中涉及的门控电荷有贡献。
