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

在过去的几年里，我们已经开发了跨膜和 Shaker，KvAP，hERG，NaChBac和Ca 2+通道的细胞外片段在静息，开放和 许多过渡构象。这些通道的分子动力学模拟嵌入在 进行脂质双层以评估和改进模型。这些模型被限制在 通过最近获得的实验数据;例如，Kv1.2通道的晶体结构， KvAP通道的电子顺磁共振（EPR）研究，热力学循环 来自蝎子的BeKM 1毒素与hERG通道结合的诱变研究，以及 半胱氨酸扫描诱变（SCAM）研究Ca 2+通道孔。我们已经证明 S4电压传感器段的电压相关运动的螺旋模型 我们在1986年首次提出，与几乎所有的实验结果一致， 能量标准，包括使用分子动力学模拟分析。最近 其他研究小组的实验和计算研究为以下方面提供了额外的支持： 我们的模特NaChBac通道是原核Na+通道，与K+具有相似性， Ca 2+和Na+通道。我们是第一个在原核生物中鉴定出这个序列的小组， 序列数据库从那时起，它就被表达出来了，它的性质也被研究 很贵目前正在努力解决其晶体结构。我们的NaChBac 使用Kv1.2通道的晶体结构作为初始模板。生成的NaChBac 模型具有涉及由P段形成的离子选择性区域的几个独特特征， 由S6段形成的激活门，以及电压感测 （S1-S4）和成孔（S5-P-S6）结构域。我们现在使用NaChBac模型作为步进 Stone来模拟更复杂的真核Ca 2+和Na+通道。到目前为止，我们已经模拟了 人和真菌Ca 2+通道的跨膜区。我们与Steffen合作 Herrings小组对这些模型进行了实验验证。具体来说，我们正在使用模型来 分析Ca 2+通道阻滞剂的分子药理学（在治疗 人类高血压和心脏病），并更好地了解突变如何与 遗传性疾病改变了Ca 2+通道的门控特性。我们已经开始了新的 项目开发HCN和TREK通道的结构和功能模型。晶体 HCN通道的环核苷酸结合结构域的结构被用于建模 胞质结构域。HCN是包括hERG通道的通道家族的成员， 我们之前已经模拟过了TREK通道是机械敏感的（牵张激活的）K+ 渠道我们正在与谢尔盖·苏哈列夫的团队合作， 开发和测试模型。我们之前与Sukharev的团队在其他方面的合作 机械敏感性通道是非常多产的。

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.