Determinants of Cardiac Repolarization

描述（由申请人提供）：人心脏中的重极化K+电流是由插入表面膜的功能通道的性质总和决定的。这些通道通常在去极化后打开，以恢复负静息电位。干扰这一过程的效率可导致延迟的肌细胞再极化。在之前的资助期内，我们确定了Kv通道KCNH2 （hERG）电流损失的新机制，并确定蛋白质加工（运输）异常是KCNH2- linked （LQT2）长QT综合征的重要机制。在本研究申请的初步数据中，我们现在表明，在我们研究的近40个人类LQT2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蛋白质运输缺陷引起的，少数突变在门控或渗透方面表现出原发性异常。我们还表明，错误加工的通道通常可以被“拯救”以发挥作用，并且存在多种拯救机制（低温、KCNH2阻断药物、thapsigargin和基因内抑制）。然而，这些救援模式是选择性的特定突变给予不同的救援模式。下一个资助期的具体目标是：1)继续验证LQT2中重极化电流的损失主要是由通道中蛋白质运输的异常引起的，并且大多数运输缺陷的LQT2突变形成的通道如果到达细胞表面膜则具有功能。2)不同2类LQT2突变的ER保留机制不同。SubAim 2a旨在鉴定WT KCNH2通道蛋白分泌途径中的不同步骤。SubAim 2b是为了验证LQT2突变在这一途径的不同步骤上破坏运输的假设。3)将KCNH2通道蛋白靶向胆固醇和鞘脂富集膜，促进了细胞膜表面的表达，改变了通道的生物物理性质。为了合理开发增加LQT2中突变KCNH2通道表面表达的新策略，需要探索调控KCNH2通道蛋白转运及其膜插入的细胞机制。

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.