Correction of Defective Protein Trafficking of a Mutant HERG Potassium Channel in Human Long QT Syndrome

Abstract

The chromosome 7-linked form of congenital long QT syndrome (LQT2) is caused by mutations in the human ether-a-go-go-related gene (HERG) that encodes the rapidly activating delayed rectifier potassium channel. One mechanism for the loss of normal channel function in LQT2 is defective protein trafficking, which results in the failure of the channel protein to reach the plasma membrane. Here we show that the N470D LQT2 mutant protein is trafficking-deficient when expressed at 37°C in HEK293 cells, whereas at 27°C its trafficking to the plasma membrane and channel function are markedly improved. We further show that the antiarrhythmic drug E-4031, which selectively blocks HERG channels, also corrects defective protein trafficking of the N470D mutant and can restore the generation of HERG current. Similar findings were obtained with the drugs astemizole and cisapride, as well as with high concentrations of glycerol. The effect of E-4031 on HERG protein trafficking was concentration-dependent and required low drug concentrations (saturation present at 5 M), developed rapidly with drug exposure, and occurred post-translationally. These findings suggest that protein misfolding leading to defective trafficking of some HERG LQT mutations may be corrected by specific pharmacological strategies. Human congenital long QT syndrome (LQT)1 is characterized by repolarization abnormalities in the heart, leading to cardiac arrhythmias, syncope, and sudden death. Defects in genes encoding pore-forming or auxiliary subunits have been identified for three types of cardiac ion channels (1-5) in human LQT. The most common defects are found in genes encoding the KVLQT1 and HERG potassium channels (1-3). For the HERG channel, which encodes the pore-forming subunit of the cardiac rapidly activating delayed rectifier potassium channel (1, 6 -8), we have recently reported that the loss of channel function can be caused by multiple mechanisms including abnormal protein trafficking, the generation of nonfunctional channels, and altered channel gating (9). In the present experiments, we studied the LQT mutation N470D heterologously expressed in a mammalian cell line. This mutation, when expressed in Xenopus oocytes, has been reported to generate HERG current with reduced amplitude (30% of wild type) and altered voltage dependence of activation (10). We found that the protein trafficking of N470D mutant is highly temperaturesensitive. In addition, we showed that the defective protein trafficking can be corrected by drugs that selectively block HERG channels. EXPERIMENTAL PROCEDURESSite-directed Mutagenesis and Transfection-The HERG N470D mutation was generated by site-directed mutagenesis of wild type HERG cDNA using the GeneEditor in vitro mutagenesis system (Promega, Madison, WI). Stable transfection of human embryonic kidney (HEK293) cells with HERG wild type and N470D cDNA was carried out using a LipofectAMINE method (11).Cell Culture and Patch Clamp Recordings-HEK293 cells were cultured at 37°C except where noted when the temperature was ...

Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

“…Western blot analysis in the cardiomyocytes showed that G601S and N470D hERG generate 135-kDa protein bands but only weak or no 155-kDa protein bands, indicating that for control conditions these two mutations generate immature protein that then does not undergo normal Golgi processing to the mature protein. E4031 has been suggested to act as a stabilizing ligand that can correct the protein folding and processing of many LQT2 trafficking-deficient hERG missense mutations, including G601S and N470D hERG, to increase surface membrane expression and I hERG (3,46). After 24 h of incubation with 10 M E4031, the 155-kDa band was increased for both G601S and N470D hERG.…”

“…For G601S and N470D hERG, the control current amplitudes were small, whereas after pharmacological correction by E4031, the amplitudes were markedly increased. In addition, N470D hERG generated outward current at the holding potential of Ϫ60 mV (see below) and had altered gating properties (34,46). In contrast, for ⌬Y475 hERG, the control I hERG amplitude was larger and culture in E4031 caused only a small increase in I hERG .…”

A Drosophila behavioral mutant, down and out ( dao ), is defective in an essential regulator of Erg potassium channels

“…Some familial forms of LQT syndrome are caused by the production of mutant hERG proteins that are inappropriately retained in the ER (26)(27)(28). Remarkably, this deficit can sometimes be overcome by treatment with certain hERGbinding drugs that somehow promote the subsequent processing and transport of the mutant channels to the plasma membrane, where they function normally (26,29,30). Dao may be an endogenous factor that functions in an essentially similar manner to promote the appearance of Erg channels at the plasma membrane.…”

Pharmacological approach to the treatment of long and short QT syndromes

“…Zhou et al (1998) first reported trafficking defects related to the expression of I Kr as a disease-causing mechanism with certain mutations associated with LQT2. Correction of these defects by culturing cells at lower temperature (27 °C) or in presence of agents like E4031, astemizole or cisapride (Zhou et al, 1999) was shown to restore function. However, most of these compounds have intrinsic HERG blocking properties which counterbalanced their corrective effects on trafficking.…”