Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
Biophys J. 2010 Nov 3;99(9):2841-52. doi: 10.1016/j.bpj.2010.08.030.
Human ether-a-go-go related gene (hERG) channel gating is associated with slow activation, yet the mechanistic basis for this is unclear. Here, we examine the effects of mutation of a unique glycine residue (G546) in the S4-S5 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by ∼-50 mV (with the exception of G546C). With the activation shift taken into account, the time constant of activation was also accelerated, suggesting a stabilization of the closed state by ∼1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the α-helical content of the S4-S5 linker suggest that the presence of G546 in wild-type hERG provides flexibility to the helix. Deactivation gating was affected differentially by the G546 substitutions. G546V induced a pronounced slow component of closing that was voltage-independent. Fluorescence measurements of voltage sensor movement in G546V revealed a slow component of voltage sensor return that was uncoupled from charge movement, suggesting a direct effect of the mutation on voltage sensor movement. These data suggest that G546 plays a critical role in channel gating and that hERG channel closing involves at least two independently modifiable reconfigurations of the voltage sensor.
人类 ether-a-go-go 相关基因 (hERG) 通道门控与缓慢激活有关，但这种机制基础尚不清楚。在这里，我们研究了突变 S4-S5 连接环中独特的甘氨酸残基 (G546) 对电压传感器运动及其与孔门控的偶联的影响。用具有不同物理化学性质的残基取代 G546，将激活门控移位约-50 mV（除 G546C 外）。考虑到激活移位，激活的时间常数也会加速，表明通过 1.6-4.3 千卡/摩尔（相当于一个到两个氢键的能量）稳定了关闭状态。S4-S5 连接环的α-螺旋含量预测表明，野生型 hERG 中的 G546 为螺旋提供了灵活性。失活动力学门控受到 G546 取代的不同影响。G546V 诱导出与电压无关的显著慢关闭成分。G546V 中电压传感器运动的荧光测量揭示了与电荷运动解耦的电压传感器返回的慢成分，表明突变对电压传感器运动有直接影响。这些数据表明，G546 在通道门控中起着关键作用，并且 hERG 通道关闭至少涉及电压传感器的两种独立可修饰的重新配置。
