PDF(Pubmed)
Abstract：
Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson-Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.
摘要：
从电压传感器到失活门的运动传输是离子通道一般生理学中的重要问题。在通道hNav1.5的低温EM结构中，胞外环IVP2-S6中的残基N1736和R1739分别接近谷氨酸E1225和E1295，在电压传感域III(VSD-III)中。ClinVar报告的变体E1230K，E1295K,和R1739W/Q和其他变体在环IVP2-S6，IIIS1-S2和IIIS3-S4与心律失常有关，强调IVP2-S6和VSD-III之间的接口是疾病突变的热点。由这些突变引起的通道功能障碍的原子机制是未知的。这里,我们产生了突变体E1295R,R1739E,E1295R/R1739E,和N1736R,在HEK-293T细胞中表达，并探索了生物物理特性。突变E1295R减少了稳态快速失活和增强的稳态缓慢失活。相比之下,突变R1739E轻微增强快速失活和减弱缓慢失活。双突变体E1295R/R1739E的特征与野生型通道的特征相当相似。突变N1736R减弱缓慢失活。分子建模预测了R1739E与激活的电压感应螺旋IIIS4中最外层赖氨酸的盐桥接。相比之下,功能缺失替代E1295R排斥R1739,从而使激活的VSD-III不稳定,这与我们的E1295R引起G-V曲线去极化偏移的数据一致.在约束维持的盐桥E1295-R1739下，VSD-III的计算机模拟失活导致以下变化：1）IIIS4和IVS5之间的接触被切换；2）接头-螺旋IIIS4-S5与IVS5，IVS6的接触和快速失活三肽IFM被修饰；3）IFM三肽与螺旋IVS5和IVS6的接触有助于缓慢的IVS6中的螺旋变化。停用的VSD-III中的盐桥E1295-R1739的可能性得到了泊松-玻尔兹曼计算和循环IVP2-S6的状态相关能量学的支持。一起来看,我们的结果表明,IVP2-S6环路参与了从VSD-III到失活门的运动传递.
