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

在过去的两年里，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，我们在1986年首次提出的S4电压传感器片段随电压变化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进行分析。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·赫林和安吉·盖利的团队合作，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，并且可能作为抗真菌剂很重要)，并更好地了解与遗传疾病相关的突变如何改变钙通道的门控特性。关于我们的NaChBac和人钙离子通道模型的手稿已经提交。我们已经启动了一个新的项目来开发具有环核苷酸结构域的通道家族[EAG和ERG，AKT(植物)，PAK(草履虫)，CNG和HCN]的结构和功能模型。在对HERG和HCN通道进行建模方面取得了实质性进展。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。我们之所以关注这一家族，是因为他们中的一些人(特别是与HERG通道密切相关的EAG通道)的表达与多种癌症有关。我们正在与Gea-Ny Tseng的实验室合作，对我们HERG通道的各个方面进行实验测试。

During the last two 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 Herring's and Angie Gelli's groups 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 potentially important as antifungicides) and to better understand how mutations associated with genetic diseases alter the gating properties of Ca2+ channels. Manuscripts have been submitted on our models of NaChBac and human Ca2+ channels. We have started a new project to develop structural and functional models of channel families [EAG and ERG, AKT (plant), PAK (paramecium), CNG, and HCN] that possess a cyclic nucleotide domain. Substantial progress has been made in modeling the hERG and HCN channels. A crystal structure of the cyclic nucleotide-binding domain of the HCN channel is being used to model the cytoplasmic domain. We are focusing on this group of families because expression of some of them (especially EAG channels that are closely related to hERG channels) has been associated with several cancers. We are collaborating with Gea-Ny Tseng's lab to experimentally test aspects of our hERG channels.