Labro Alain J, Grottesi Alessandro, Sansom Mark S P, Raes Adam L, Snyders Dirk J
Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Am J Physiol Cell Physiol. 2008 Jun;294(6):C1476-84. doi: 10.1152/ajpcell.00479.2007. Epub 2008 Apr 2.
The Kv1-4 families of K+ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently.
Kv1 - 4家族的钾离子通道在S6螺旋中含有串联脯氨酸基序（PXP），这对通道门控至关重要。在人类Kv1.5中，将第一个脯氨酸替换为丙氨酸会导致通道失去功能。通过在附近位置引入另一个脯氨酸将序列变为AVPP，该突变体得以挽救。这产生了一种通道，在第一次去极化时能快速激活（毫秒范围）。然而，此后，该通道被困在另一种门控模式中，其特征是激活动力学缓慢（秒范围）且电压依赖性较浅。即使在非常短的去极化步骤中也观察到了门控模式的转换，但恢复到初始的“快速”模式极其缓慢。计算模型表明转换发生在通道失活期间。为了测试改变后的PXP序列对S6螺旋移动性的影响，我们对野生型（WT）和突变体的分离S6结构域进行了分子动力学模拟，模拟从关闭或开放构象开始。WT S6螺旋在从任一状态开始的模拟中都在PXP区域周围显示出移动。然而，具有AVPP序列的S6仅在从关闭构象开始时显示出灵活性，而从开放状态开始时则是刚性的。这些结果表明，PXP基序周围的区域可能充当“铰链”，将序列改变为AVPP会导致通道失活至一种具有交替构象的状态，使其随后“不愿意”开放。
