Determinants of Cardiac Repolarization

描述（申请人提供）：人心脏中的复极化K+电流由插入表面膜的功能通道的性质之和控制。这些通道通常在去极化后打开以恢复负静息电位。这一过程效率的紊乱可导致延迟的肌细胞复极。在上一个资助期，我们确定了Kv通道KCNH2（hERG）电流丢失的新机制，并确定了蛋白质加工（运输）异常是KCNH2相关（LQT2）长QT综合征的重要机制。在这项研究应用的初步数据中，我们现在表明，在我们研究的近40种人类LQT 2突变中，蛋白质运输缺陷是常见的。因此，LQT2中K+电流表型的丧失主要是由缺陷性蛋白质运输引起的，少数突变显示门控或渗透的主要异常。我们还发现，错误处理的通道通常可以被“拯救”以发挥功能，并且存在多种拯救机制（低温，KCNH2阻断药物，毒胡萝卜素和基因内抑制）。然而，这些拯救模式对特定突变具有选择性，从而产生不同的拯救模式。下一个资助期的具体目标是：1）继续检验以下假设：LQT 2中复极化电流的丧失主要是由通道的蛋白质运输异常引起的，并且大多数运输缺陷的LQT 2突变形成的通道如果到达细胞表面膜则是功能性的。2)不同2类LQT2突变的ER保留通过不同的机制发生。SubAim 2a旨在确定WT KCNH2通道蛋白分泌途径中的不同步骤。SubAim 2b旨在检验LQT 2突变在该途径的不同步骤破坏运输的假设。3)将KCNH2通道蛋白靶向到富含胆固醇和鞘脂的膜中促进在细胞表面膜处的表达并改变通道的生物物理性质。为了合理地开发新的策略，增加突变的KCNH2通道在LQT 2的表面表达，调节KCNH2通道蛋白的运输和膜插入的细胞机制需要探索。

DESCRIPTION (provided by applicant): Repolarizing K+ current in human heart is governed by the sum of properties of function channels inserted into the surface membrane. These channels normally open following depolarization to restore a negative resting potential. Disturbances in the efficiency of this process can lead to delayed myocyte repolarization. In the previous grant period, we identified novel mechanisms for loss of current of the Kv channel KCNH2 (hERG), and identified protein processing (trafficking) abnormalities as an important mechanism in KCNH2- linked (LQT2) long QT syndrome. In preliminary data in this research application, we now show that of nearly 40 human LQT2 mutations we have studied functionally, protein trafficking defects are common. Thus, the loss of K+ current phenotype in LQT2 is dominated by defective protein trafficking, with a small number of mutations showing primary abnormalities in gating or permeation. We also showed that mis-processed channels often can be "rescued" to function and that multiple mechanisms for rescue exist (low temperature, KCNH2 blocking drugs, thapsigargin, and intragenic suppression). These rescue modalities, however, are selective for specific mutations giving different patterns of rescue. The specific aims for the next grant period are: 1) To continue to test the hypothesis that the loss of repolarizing current in LQT2 is caused predominately by abnormalities in protein trafficking of the channels and that most trafficking-deficient LQT2 mutations form channels that are functional if they reach the cell surface membrane. 2) ER retention of different Class 2 LQT2 mutations occurs by distinct mechanisms. SubAim 2a is to identify distinct steps in the secretory pathway for WT KCNH2 channel protein. SubAim 2b is to test the hypothesis that LQT2 mutations disrupt trafficking at distinct steps in this pathway. 3) Targeting KCNH2 channel proteins into cholesterol and sphingolipid enriched membranes facilitates expression at the cell surface membrane and modifies the biophysical properties of channels. In order to rationally develop new strategies that increase surface expression of mutated KCNH2 channels in LQT2, the cellular mechanisms that regulate the trafficking of KCNH2 channel proteins and their membrane insertion need to be explored.