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

我研究的主要主题是描述导致HCN和HERG(KCNH2)离子通道截然不同的生理特征的结构-功能关系。我们利用了这样一个事实，即它们具有不同的氨基酸序列相似和不同的结构域。我们的实验方法将通道的三级结构及其嵌合体的电子计算机模型链接到膜片钳功能的评估。目的是了解它们渗透路径、离子选择性和门控特性不同的结构决定因素。 对于钾离子通道，孔衬序列(S/T)(V/I)G(Y/F)G在K+选择性中起着关键作用。Backx实验室通过将HCN4选择性过滤器(CIGYG)附近的突变替换恢复到钾通道(SIGYG)中看到的方式，检查了突变替换的影响。他们的发现表明，HCN4选择性过滤器对SIGYG K+通道序列的突变不足以赋予HCN4对K+的选择性。他们得出结论，HCN4和K+通道选择性的差异源于P环外的差异。HCN和HERG的选通特性也有明显的不同。HCN编码在超极化电位下被激活的通道并诱导内向去极化电流，而HERG电流被去极化电位激活并诱导外向再极化电流。 有关离子通道的结构和功能信息呈爆炸性增长。有趣的是，最近的报道显示，Shaker K+通道电压传感器跨膜片段中的一个突变体(S4-R1-Ser)支持通过电压传感器片段的另一种离子渗透途径，而不需要通过传统的选择性过滤器。这些渗透路径被称为“欧米伽”流。我们发现，HERG(K525S(Ser))电压传感器的单点突变产生了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HCN4非常相似。尽管如此，K525S Herg和HCN4在锂选择性和微摩尔铯对HCN4的药理阻断方面仍存在未解决的差异。最重要的是，我们只有间接证据表明，HCN是欧米伽电流的结果。我们的目的是将原子学模型与膜片钳研究联系起来，以深入了解产生omega电流的拓扑重排及其与HCN4电流的生理相关性。实验上，我们的方法用于计算机设计的嵌合体，以及结构-功能的系统发育方法。Omega电流在正常生理中的作用对调节动作电位的形态、自律性和同步性具有潜在的重要意义。

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.