基于孔道突变的钙库控制钙离子通道的门控机制研究

钙释放激活的钙通道(CRAC)是非兴奋性细胞中钙离子内流的主要途径. 然而，CRAC激活过程受到多种因素的调控，研究的主要困难在于该通道蛋白的孔结构Orai及其激活蛋白STIM复合体的结构复杂，难以在原子尺度上通过实验观察到完整的激活及失活过程. 电生理和生化实验表明，与野生型Orai孔道的开启需要STIM蛋白激活不同，Orai蛋白上某些氨基酸的突变会导致其变成自发开启的状态. 因此，研究者普遍使用处于自发开启状态的通道突变体，通过研究突变体的结构和电生理性质，推测野生型孔道在生理条件下可能的激活机制，并提出了不同的调控机制模型(Biopolymers 111，e23392 (2020)). 然而，突变体是否可以真实地反映CRAC通道的激活机理还存在一些不确定性. 本文采用微秒级分子动力学模拟，结合自由能计算和基于电扩散连续模型的有限元分析，系统性地探索了三种处于激活状态的dOrai突变体(G170P、H206A和P288A)的结构及动态变化、离子通过自由能及电流电压曲线，并与以往工作中对于其它突变体(Proc. Natl. Acad. Sci. USA 110，17332 (2013)；J. Phys. Chem. B 118，9668 (2014))以及野生型处于开启状态的一种可能的结构模型(iScience 16，356 (2019))进行比较. 计算结果表明，三种突变体的孔道结构均呈现出扩张的趋势，孔道内水分子的有序排布程度降低，从而显著降低了阳离子在孔道内扩散的自由能. 但不同的dOrai突变体采用了不同的模式来保持其通道处于激活状态. 具体的说，G170P突变体通过引入脯氨酸使直接形成孔道的跨膜螺旋TM1发生显著弯曲，从而有效增大孔径. H206A和P288A突变体都是通过别构效应间接地改变孔道结构：其中H206A 通过在TM1外侧的跨膜螺旋TM2上引入侧链较小的丙氨酸而破坏同一条链上TM1和TM2间的堆积作用，引起TM1的扩张；而P288A的突变位点处于孔道蛋白最外侧的跨膜螺旋TM4上，减小TM4的弯曲程度，并进一步将构象变化传递到孔道中心的TM1，从而使得孔径增大而激活通道. 因此，虽然三个突变体均可以导致Orai孔道的自发激活，但突变位点各不相同，导致构象变化的分子机制也各有差异. 此外，与野生型dOrai通道处于激活状态的结构模型相比，上述突变体的孔道结构、整流性质和阳离子选择性都有明显差异. 因此，对于突变体的研究可以为理解Orai 通道的激活机制提供信息，但并不能真实地反映生理条件下由激活蛋白STIM调控的Orai通道的门控过程.

Mutants only Partially Represent Characteristics of Calcium-Release-Activated Calcium Channel Gating

Calcium-release-activated calcium (CARC) channels are one of the major pathways of cal-cium entry in non-excitable cells. Despite a decade or two of research, its regulatory mech-anism is not yet thoroughly understood. The slow progress is due to the complexity of its pores (i.e., Orai) on one hand and the diffi-culty in capturing its regulatory complex on the other hand. As a result, possible gating mechanisms have often been speculated by ex-ploring the structure and properties of consti-tutive open mutants. However, there is much debate about how they can truly reflect the gating of CRAC channels under physiological conditions. In the present study, we combined molecular dynamics simulations with free energy calculations to study three dOrai mutants (G170P, H206A, and P288A), and further calculated their current-voltage curves. Results show that these constructs adopt different approaches to maintain their conductive state. Meanwhile they have unique pore structures and distinctive recti cation properties and ion selectivity for cations compared to wild-type pores. We conclude that although the mutants may partially capture the gating motion characteristics of wild-type pores, the information obtained from these mutants is likely not a true reflection of CRAC channel gating under physiological conditions.