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

电压门控通道的结构和功能机制建模

• 批准号: 8157412
• 负责人: HOMER ROBERT GUY
• 金额: $ 38万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157412
• 关键词: Modeling; Structure; voltage gated channel

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.

在过去的几年中，我们开发了Shaker、KvAP、hERG、NaChBac和Ca2+通道在静息、开放和许多过渡构象中的跨膜和细胞外片段的结构模型。这些通道嵌入在脂质双分子层的分子动力学模拟进行了评估和完善的模型。模型受新近获得的实验数据的约束；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诱变研究，以及Ca2+通道孔的半胱氨酸扫描诱变（SCAM）研究。我们已经证明，我们在1986年首次提出的S4电压传感器段的电压依赖运动的螺旋螺旋模型几乎与所有的实验结果和能量标准一致，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Na+通道，与K+、Ca2+和Na+通道相似。我们是第一个在原核生物序列数据库中发现这个序列的小组。从那时起，人们对它进行了表达，并对它的性质进行了昂贵的研究。人们正在努力解开它的晶体结构。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。所得到的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段形成的激活门，以及电压感应（S1-S4）和孔隙形成（S5-P-S6）域之间的相互作用。我们现在使用NaChBac模型作为跳板来模拟更复杂的真核Ca2+和Na+通道。到目前为止，我们已经模拟了人类和真菌Ca2+通道的跨膜区域。我们正在与斯蒂芬·赫林团队合作，对这些模型进行实验测试。具体来说，我们正在使用这些模型来分析Ca2+通道阻滞剂的分子药理学（在治疗人类高血压和心脏病中很重要），并更好地了解与遗传疾病相关的突变如何改变Ca2+通道的门控特性。启动HCN、TREK频道结构和功能模式建设新项目。HCN通道环核苷酸结合域的晶体结构被用来模拟细胞质域。HCN是一个通道族的成员，其中包括我们之前建模的hERG通道。TREK通道是机械敏感（拉伸激活）K+通道。我们正在与Sergei Sukharev的团队合作开发和测试这些模型。我们之前与Sukharev的团队在其他机械敏感渠道上的合作非常有成效。