Objective To identify the cellular proteins which interact with HERG channel and to study the effects of these protein interaction on the HERG channel function. Methods: (1) Yeast two-hybrid screen. Construct the bait plasmid pGBKT7-herg-NT. The AH109 transformed with pGBKT7-herg-NT was mated with a pre-transformed human heart cDNA library and then the mating mixture was grown on the selected medium. The prey plasmids were isolated from the putative positive colonies, and then were sequenced. (2) To further confirm the interaction, the Co-immunoprecipitation was performed by using specifi c antibody. (3) GST pull-down assay. To study the PTPN12-HERG interaction, the GST-HERG- NT fusion protein was expressed and purifi ed, then the proteins pulled down by the GST-HERG-NT was Western blot analyzed using anti-PTPN12 antibody. (4) Immuno? uorescence analysis. To study the co-localization between PTPN12 and HERG, the anti-PTPN12 antibody and anti-HERG antibody were used to probe the subcellular localization of these two proteins. Results: (1) In the yeast two-hybrid screening using the HERG-NT as the bait, Protein tyrosine phosphatase nonreceptor type 12 (PTPN12) was identifi ed as the potential HERG partner. (2) Co-immunoprecipitation. The anti-HERG antibody precipitated the PTPN12 and the HERG complex from the rat heart lysates. (3) GST pull-down assay. The GST-HERG-NT fusion protein, but not the GST protein, pulled the PTPN12 down from the rat heart lysates. (4) Immunofluorescence analysis. The co-localization of the PTPN12 and HERG occurred mostly in the membrane surface compartment, where the majority of HERG present. Conclusions: PTPN12 interacts with HERG potassium channel in cardiac cell. This novel fi nding may help to further understand the molecular basis of HERG channel diversity and arrhythmogenesis in the long-QT syndrome.

Protein-protein interaction plays a key role in the regulation of biological processes. The human potassium (HERG) channel is encoded by the ether-à-go-go-related gene (herg), and its activity may be regulated by association with other cellular proteins. To identify cellular proteins that might play a role in the regulation of the HERG channel, we screened a human heart cDNA library with the N terminus of HERG using a yeast 2-hybrid system, and identified caveolin-1 as a potential HERG partner. The interaction between these 2 proteins was confirmed by coimmunoprecipitation assay, and their overlapping subcellular localization was demonstrated by fluorescence immunocytochemistry. The physiologic implication of the protein-protein interaction was studied in whole-cell patch-clamp electrophysiology experiments. A significant increase in HERG current amplitude and a faster deactivation of tail current were observed in HEK293/HERG cells in a membrane lipid rafts disruption model and caveolin-1 knocked down cells by RNA interference. Alternatively, when caveolin-1 was overexpressed, the HERG current amplitude was significantly reduced and the tail current was deactivated more slowly. Taken together, these data indicate that HERG channels interact with caveolin-1 and are negatively regulated by this interaction. The finding from this study clearly demonstrates the regulatory role of caveolin-1 on HERG channels, and may help to understand biochemical events leading to arrhythmogenesis in the long QT syndrome in cardiac patients.

Human ether-a-go-go-related gene(HERG),which was identified from the cDNA library of human hippocampus,has homology with the EAG gene in the Drosophila.HERG has now been shown to encode the α subunit of the delayed rectification potassium channel that is belong to the voltage-dependent potassium channel family.Researchers have determined that the changes in the structures and functions of the channel,caused by a gene mutation of HERG,alter the action potential duration of the myocardial cells,thus displaying the manifestation of arrhythmia.Nowadays,it has been that HERG has something to do with the onset of arrhythmia,especially the long QT syndrome 2.This article provides an overview of the structure of the gene and the channel,the property of this potassium channel and its connection with the occurrence of arrhythmia.

Protein-protein interactions are critical for protein trafficking, localization and the regulation of ion channels. The human ether-a-go-go-related gene (herg) encodes the alpha-subunit of the potassium channel underlying the rapid component of the cardiac delayed rectifier current. To identify the cellular proteins involved in the regulation of the HERG channel, a human heart cDNA library was screened using a yeast two-hybrid system, with the N-terminus of HERG as bait. The four and a half LIM domain protein 2 (FHL2) was identified as a potential HERG partner. The interaction between these two proteins was confirmed by co-immunoprecipitation and glutathione transferase pull-down assays and immunocytochemical analysis. The physiological implication of HERG-FHL2 interaction, assessed by whole-cell, patch-clamp electrophysiology experiments, showed a significant increase in the HERG current amplitude and a faster deactivation of the tail current in human embryonic kidney 293 cells co-expressing HERG and FHL2. These data indicate that FHL2 interacts with and regulates the HERG channel. Our findings may aid in the further understanding of the molecular basis of HERG channel diversity and arrhythmogenesis in the long-QT syndrome.