Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
J Gen Physiol. 2013 Sep;142(3):289-303. doi: 10.1085/jgp.201310992.
Human ether-à-go-go-related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance-voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed-open equilibria, similar to that proposed for F290 in Shaker channels.
人类 ether-à-go-go 相关基因 (hERG, Kv11.1) 钾通道的激活和失活动力学非常缓慢。有人提出，在快速激活的 Shaker 通道中，S2 片段中高度保守的 Phe 残基 (F290) 形成了一个假定的门控电荷转移中心，与 S4 门控电荷相互作用，即 R362 (R1) 和 K374 (K5)，并催化它们穿过聚焦电场的运动。F290 在 hERG 中保守 (F463)，但 hERG S4 中的相关残基是反转的，即 K525 (K1) 和 R537 (R5)，并且在 R537 旁边有一个额外的正电荷 (即 K538)。我们已经研究了 hERG 通道是否具有类似于 Shaker 中描述的转移中心，如果这些 S4 电荷差异导致 hERG 通道的门控缓慢。在测试的五个 hERG F463 疏水性取代中，F463W 和 F463Y 将电导-电压 (G-V) 关系移向更去极化的电位，并显著减慢通道激活。考虑到 S4 残基的反转 (即 K525、R537)，F463W 对关闭状态的稳定作用与 F290 在 Shaker 中的作用相似。与 Shaker 的结果预测一致，hERG K525R 突变使关闭状态不稳定。然而，hERG R537K 并没有像预测的那样稳定开放状态。相反，我们发现相邻的 K538 残基对于开放状态的稳定至关重要，因为 K538R 显著减慢并右移了激活的电压依赖性。最后，对 F463W/K525R 和 F463W/K538R 双突变的 G-V 曲线进行双突变循环分析表明，F463 与 S4 片段中的 K525 和 K538 形成功能相互作用。总的来说，这些数据表明 F463 在介导关闭-开放平衡中起作用，类似于 Shaker 通道中提出的 F290 的作用。
