Nakajo Koichi, Kubo Yoshihiro
Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan.
J Gen Physiol. 2007 Sep;130(3):269-81. doi: 10.1085/jgp.200709805. Epub 2007 Aug 13.
KCNQ1 is a voltage-dependent K(+) channel whose gating properties are dramatically altered by association with auxiliary KCNE proteins. For example, KCNE1, which is mainly expressed in heart and inner ear, markedly slows the activation kinetics of KCNQ1. Whether the voltage-sensing S4 segment moves differently in the presence of KCNE1 is not yet known, however. To address that question, we systematically introduced cysteine mutations, one at a time, into the first half of the S4 segment of human KCNQ1. A226C was found out as the most suited mutant for a methanethiosulfonate (MTS) accessibility analysis because it is located at the N-terminal end of S4 segment and its current was stable with repetitive stimuli in the absence of MTS reagent. MTS accessibility analysis revealed that the apparent second order rate constant for modification of the A226C mutant was state dependent, with faster modification during depolarization, and was 13 times slower in the presence of KCNE1 than in its absence. In the presence of KCNE3, on the other hand, the second order rate constant for modification was not state dependent, indicating that the C226 residue was always exposed to the extracellular milieu, even at the resting membrane potential. Taken together, these results suggest that KCNE1 stabilizes the S4 segment in the resting state and slows the rate of transition to the active state, while KCNE3 stabilizes the S4 segment in the active state. These results offer new insight into the mechanism of KCNQ1 channel modulation by KCNE1 and KCNE3.
KCNQ1是一种电压依赖性钾离子通道，其门控特性会因与辅助性KCNE蛋白结合而发生显著改变。例如，主要在心脏和内耳中表达的KCNE1会显著减慢KCNQ1的激活动力学。然而，在KCNE1存在的情况下，电压感应S4段的移动是否不同尚不清楚。为了解决这个问题，我们将半胱氨酸突变逐个系统地引入人KCNQ1的S4段前半部分。发现A226C是最适合进行甲硫基磺酸盐（MTS）可及性分析的突变体，因为它位于S4段的N端，并且在没有MTS试剂的情况下，其电流在重复刺激下是稳定的。MTS可及性分析表明，A226C突变体修饰的表观二级速率常数取决于状态，在去极化过程中修饰更快，并且在KCNE1存在时比不存在时慢13倍。另一方面，在KCNE3存在的情况下，修饰的二级速率常数不依赖于状态，这表明即使在静息膜电位下，C226残基也总是暴露于细胞外环境。综上所述，这些结果表明，KCNE1使S4段在静息状态下稳定，并减慢向激活状态的转变速率，而KCNE3使S4段在激活状态下稳定。这些结果为KCNE1和KCNE3对KCNQ1通道的调节机制提供了新的见解。
