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.
Sub Aim 2 a is to identify distinct steps in the secretory pathway for WT KCNH2 channel protein.
Sub Aim 2 b 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.
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