Mapping of the structure - function role of important selected ion channels for normal physiology

正常生理学中重要选定离子通道的结构-功能作用图谱

• 批准号: RGPIN-2016-04066
• 负责人: Duff, Henry
• 金额: $ 2.26万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614817
• 关键词: Mapping; structure; function; role; important

The overarching theme of my research is to characterize the structure-function relationships responsible for the distinctly different physiologic characteristics of HCN and hERG (KCNH2) ion channels. We take advantage of the fact that they have domains of distinct amino acid sequence similarities and differences. Our experimental approach links in silico models of the tertiary structure of the channels and their chimeras to patch clamp assessment of function. The purpose is to understand the structural determinants of differences in their permeation paths, ion selectivity and gating characteristics. For potassium channels, the pore-lining sequence (S/T)(V/I)G(Y/F)G plays a pivot role in K+-selectivity. The Backx laboratory examined the impact of mutation substitutions near the selectivity filter of HCN4 (CIGYG) by reverting it to that seen in potassium channels (SIGYG). Their findings indicated that mutations of the selectivity filter of HCN4 to the SIGYG K+ channel sequence was insufficient to confer K+ selectivity to HCN4. They concluded that differences in selectivity of HCN4 versus K+ channels originated from differences outside the P-loop. The gating characteristics of HCN and hERG are also distinctly different. HCN encodes a channel that is activated at hyperpolarizing potentials and induces an inward depolarizing current, whereas the hERG current is activated by depolarized potentials and induces an outward repolarizing current. There has been an explosive growth of structural and functional information about ion channels. Intriguingly, recent reports show that a mutant (S4-R1-Ser) in the voltage-sensor transmembrane segments of the Shaker K+ channel supports an alternative ion-permeation pathway via the voltage-sensor segments without flux through the conventional selectivity filter. These permeation pathways are termed “omega” currents. We discovered that a single point mutation in the voltage-sensor of hERG (K525S (Ser)) generates a dofetilide-insensitive inward current elicited by hyperpolarization with features quite similar to wild-type HCN4. Even so, there are unresolved differences between K525S hERG and HCN4 including lithium selectivity and pharmacologic block of HCN4 by µMol cesium. Most importantly, we only have circumstantial evidence that HCN is a consequence of an omega current. Our intent is to link atomistic models to patch clamp studies to provide an in-depth understand the topologic rearrangements generating omega currents and their physiologic relevance to HCN4 currents. Experimentally our approach uses in silico designed chimeras, and phylogenetic approach to structure-function. The emerging role of omega currents in normal physiology is potential important to regulate action potential shape, automaticity and synchronicity.

我的研究的首要主题是表征HCN和hERG（KCNH 2）离子通道具有明显不同生理特征的结构-功能关系。我们利用它们具有不同氨基酸序列相似性和差异性的结构域的事实。我们的实验方法将通道的三级结构及其嵌合体的计算机模型与膜片钳功能评估联系起来。其目的是了解其渗透路径，离子选择性和门控特性的差异的结构决定因素。 对于钾通道，孔衬序列（S/T）（V/I）G（Y/F）G在K+选择性中起关键作用。Backx实验室通过将HCN 4（CIGYG）的选择性过滤器恢复为钾通道（SIGYG）中所见的突变取代来检查其影响。他们的研究结果表明，HCN 4的选择性过滤器到SIGYG K+通道序列的突变不足以赋予HCN 4 K+选择性。他们得出结论，HCN 4与K+通道的选择性差异源于P环外的差异。HCN和hERG的门控特性也明显不同。HCN编码在超极化电位下激活并诱导向内去极化电流的通道，而hERG电流由去极化电位激活并诱导向外复极化电流。 关于离子通道的结构和功能的信息已经有了爆炸性的增长。有趣的是，最近的报告表明，突变体（S4-R1-Ser）的电压传感器跨膜段的振动钾离子通道支持一个替代的离子渗透途径，通过电压传感器段没有流量通过传统的选择性过滤器。这些渗透途径被称为“Ω”电流。我们发现hERG电压传感器中的单点突变（K525 S（Ser））产生由超极化引起的多非利特不敏感的内向电流，其特征与野生型HCN 4非常相似。即便如此，K525 S hERG和HCN 4之间仍存在未解决的差异，包括锂的选择性和μMol铯对HCN 4的药理学阻断。最重要的是，我们只有间接证据表明HCN是欧米茄电流的结果。我们的目的是将原子模型与膜片钳研究联系起来，以深入了解产生Ω电流的拓扑重排及其与HCN 4电流的生理相关性。在实验上，我们的方法使用计算机设计的嵌合体，和系统发育方法的结构-功能。欧米茄电流在正常生理学中的新兴作用对于调节动作电位形状、自动性和同步性具有潜在的重要性。