Modeling of the structure and functional mechanisms of voltage-gated channels

在过去的几年里，我们已经建立了Shaker、KvAP、HERG、NaChBac和钙离子通道在静止、开放和大量过渡构象中的跨膜和胞外片段的结构模型。对嵌入在脂质双层中的这些通道进行了分子动力学模拟，以评估和改进模型。这些模型受到最近获得的实验数据的限制；例如，Kv1.2通道的晶体结构，KvAP通道的电子顺磁共振(EPR)研究，蝎子BeKM1毒素与HERG通道结合的热力学循环突变研究，以及钙通道孔的半胱氨酸扫描突变(SCAM)研究。我们已经证明了我们在1986年首次提出的S4电压传感器片段的电压依赖运动的螺旋模型与几乎所有的实验结果和能量标准是一致的，包括使用分子动力学模拟的分析。最近来自其他小组的实验和计算研究为我们的模型提供了额外的支持。NaChBac通道是一种原核生物Na+通道，与K+、Ca~(2+)和Na~+通道有相似之处。我们是第一个在原核序列数据库中鉴定该序列的小组。从那时起，它得到了表达，其性质也得到了昂贵的研究。目前正在努力解决其晶体结构问题。我们的NaChBac是使用Kv1.2通道的晶体结构作为初始模板开发的。得到的NaChBac模型具有几个独特的特征，包括由P段形成的离子选择区，由S6段形成的活化门，以及电压敏感(S1-S4)和成孔(S5-P-S6)结构域之间的相互作用。我们现在正在使用NaChBac模型作为踏脚石来模拟更复杂的真核细胞的钙离子和钠离子通道。到目前为止，我们已经对人和真菌的钙离子通道的跨膜区进行了建模。我们正在与斯特芬鲱鱼集团合作，对这些模型进行实验测试。具体地说，我们正在使用这些模型来分析钙通道阻滞剂的分子药理学(在治疗人类高血压和心脏病方面很重要)，并更好地了解与遗传病相关的突变如何改变钙通道的门控特性。我们已经启动了新的项目，以开发HCN和Trek通道的结构和功能模型。HCN通道的环核苷酸结合域的晶体结构正被用来模拟细胞质结构域。HCN是包括HERG通道的通道家族的成员，我们之前已经对其进行了建模。Trek通道是机械敏感(拉伸激活)的K+通道。我们正在与谢尔盖·苏哈雷夫在大学模型中的团队合作开发和测试这些模型。我们之前与苏哈雷夫的团队在其他机械敏感渠道上的合作非常富有成效。

During the last few years, we have developed structural models of the transmembrane and extracellular segments of Shaker, KvAP, hERG, NaChBac, and Ca2+ channels in resting, open, and numerous transition conformations. Molecular dynamic simulations of these channels embedded in a lipid bilayer were performed to evaluate and refine the models. The models were constrained by recently obtained experimental data; e.g., the crystal structure of the Kv1.2 channel, electron paramagnetic resonance (EPR) studies of KvAP channels, thermodynamic cyclic mutagenesis studies of the binding of BeKM1 toxin from scorpions to the hERG channel, and cysteine scanning mutagenesis (SCAM) studies of Ca2+ channel pores. We have demonstrated that the helical screw model for the voltage-dependent movement of the S4 voltage-sensor segment that we proposed first in 1986, is consistent with virtually all experimental results and energetic criteria, including analyses using molecular dynamic simulations. Recent experimental and computational studies from other groups have provided additional support for our models. The NaChBac channel is a prokaryotic Na+ channel that has similarities to K+, Ca2+, and Na+ channels. We were the first group to identify this sequence in the prokaryotic sequence data base. Since then, it has been expressed and its properties have been studied expensively. Efforts are underway to solve its crystal structure. Our NaChBac was develop using the crystal structure of the Kv1.2 channel as an initial template. The resulting NaChBac model has several unique features involving the ion selective region formed by the P segments, the activation gate formed by the S6 segment, and the interaction between the voltage-sensing (S1-S4) and pore-forming (S5-P-S6) domains. We are now using the NaChBac model as a stepping stone to model more complex eukaryotic Ca2+ and Na+ channels. So far we have modeled the transmembrane regions of human and fungal Ca2+ channels. We are collaborating with Steffen Herrings group to test experimentally these models. Specifically, we are using the models to analyze the molecular pharmacology of Ca2+ channel blockers (important in treating hypertension and heart disease in humans) and to better understand how mutations associated with genetic diseases alter the gating properties of Ca2+ channels. We have started new projects to develop structural and functional models of HCN and TREK channels. A crystal structure of the cyclic nucleotide-binding domain of the HCN channel is being used to model the cytoplasmic domain. HCN is a member of a channel family that includes the hERG channel, which we have modeled previously. TREK channels are mechanosensitive (stretch-activated) K+ channels. We are collaborating with Sergei Sukharev's group at the University model in developing and testing the models. Our previous collaborations with Sukharev's group on other mechanosensitive channels were very productive.