STRUCTURE-FUNCTION RELATION & MODULATION OF Kv CHANNELS

描述(申请人提供)：该项目有三个相互关联的目标：(1)提供心脏主要电压门控钾(Kv)通道功能的结构信息，(2)了解Kv通道成分突变导致功能丧失或获得的原因，以及(3)确定针对心脏Kv通道的新治疗策略。该提案的重点是慢速延迟整流(IKS)通道。IKS有两个主要成分：形成孔道的KCNQ1通道和辅助的KCNE1亚基。在人的心室中，IKs起着“复极储备”的作用：对b-肾上腺素能刺激作出反应时，IKs增加其电流幅度，以防止动作电位时程的过度延长。在人的心房中，IKs可能是房颤的易感因素。已发现8个与家族性房颤有关的“功能增益”KCNQ1突变。更重要的是，有报道称瓣膜疾病导致获得性房颤的KCNE1上调，这表明在这些情况下IKs的增加可能导致动作电位时程缩短和房颤持续。我们已经证明，在人类心脏中表达的另一种KCNE亚基KCNE2与KCNQ1和KCNE1在成人心肌细胞中共存。它可以与IKS通道结合形成KCNQ1/KCNE1/KCNE2三元复合体。KCNE2可在不影响门控动力学的情况下降低Iks电流幅度。家族性房颤相关突变R27C的发现突显了KCNE2作为IKS调节因子的重要性，该突变否定了KCNE2目前对IKS的抑制作用。KCNE_1和KCNE_2对心脏IKs电流幅度的调节关系尚不清楚。它们对KCNQ1通道功能的不同作用机制(S)也不是很明显。该项目旨在解决这些问题。我们是三个具有互补专业知识的研究小组(曾氏海峡生物物理、CUI计算模拟和Tian-核磁共振)，共同努力实现以下具体目标。目标1 目的是确定KCNQ1通道中跨膜螺旋(TMH)的堆积和与门控相关的运动。目的2确定KCNE1结合对KCNQ1通道中TMH相互作用的影响，以及KCNE1和KCNQ1之间的联系。目的3是确定KCNE2和KCNQ1之间的接触以及这种接触的状态依赖性。在这些实验中确定的空间关系将被用来约束KCNQ1同源模型在打开和关闭状态下。我们还将把改进后的KCNE核磁共振结构以与实验数据一致的方式对接到KCNQ1同源模型。最后，我们将测试膜通透性KCNE2模拟肽是否可以破坏KCNQ1/KCNE2相互作用并增加心肌细胞Iks电流的幅度(目标4)。这可能为深入了解KCNE1和KCNE2在IKS幅度调节方面的关系提供依据。它也是靶向KCNQ1/KCNE相互作用的治疗性多肽的原型。

DESCRIPTION (provided by applicant): This project has three interrelated objectives: (1) to provide structural information for the function of major voltage-gated potassium (Kv) channels in the heart, (2) to understand why mutations in Kv channel components lead to loss- or gain-of-function, and (3) to identify novel therapeutic strategies targeting cardiac Kv channels. The focus of this proposal is the slow delayed rectifier (IKs) channel. IKs has 2 major components: pore-forming KCNQ1 channel and auxiliary KCNE1 subunits. In human ventricles, IKs functions as a 'repolarization reserve': in response to b-adrenergic stimulation IKs increases its current amplitude to prevent excessive prolongation of action potential duration (APD). In human atria, IKs may be a liability factor for atrial fibrillation (AF). Eight 'gain-of-function' KCNQ1 mutations have been identified that are linked to familial AF. More importantly, KCNE1 upregulation has been reported for acquired AF due to valvular diseases, suggesting an increase in IKs under these conditions that can contribute to APD shortening and AF perpetuation. We have shown that another KCNE subunit expressed in human heart, KCNE2, is colocalized with KCNQ1 & KCNE1 in adult cardiac myocytes. It can associate with the IKs channel to form a KCNQ1/KCNE1/KCNE2 ternary complex. KCNE2 reduces the IKs current amplitude without affecting its gating kinetics. The importance of KCNE2 as a IKs regulator is highlighted by the identification of a familial AF-related mutation, R27C that negates the current suppressing effect of KCNE2 on IKs. The relationship between KCNE1 & KCNE2 in terms of their regulation of the IKs current amplitude in the heart is not clear. Nor is the mechanism(s) underlying their distinctly different effects on the KCNQ1 channel function. This project is designed to address these issues. We are 3 research groups with complementary expertise (Tseng - channel biophysics, Cui - computational modeling, and Tian - NMR) making a concerted effort to accomplish the following Specific Aims. Aim 1 is to determine the packing and gating-associated movements of transmembrane helices (TMHs) in the KCNQ1 channel. Aim 2 is to determine the impact of KCNE1 association on the TMH interactions in the KCNQ1 channel, and the contacts between KCNE1 & KCNQ1. Aim 3 is to determine the contacts between KCNE2 & KCNQ1 and the state-dependence of such contacts. Spatial relationships determined in these experiments will be used to constrain KCNQ1 homology models in open & closed states. We will also dock the KCNE NMR structures, after refinement, to the KCNQ1 homology models in a manner consistent with experimental data. Finally, we will test whether membrane permeable KCNE2-mimetic peptides can disrupt KCNQ1/KCNE2 interactions and increase the IKs current amplitude in cardiac myocytes (Aim 4). This could provide insights into the relationship between KCNE1 & KCNE2 in terms of IKs amplitude regulation. It also serves as a prototype for therapeutic peptides targeting KCNQ1/KCNE interactions.