Protein Tyrosine Phosphatase Nonreceptor Type 12 Interacts With And Regulates HERG Potassium Channel

Background:Encoded by the Human ether-a-go-go-related gene (HERG), HERG channel belongs to the voltage-gated channels and mediates the rapid component of the cardiac delayed rectifier current (Ikr).HERG channel underlies the regular cardiac electric activity. Mutations in the HERG gene and the blockade of the HERG-coded potassium channel by drugs cause the long QT syndrome. HERG channel underlies the regular cardiac electric activity. Mutations in the HERG gene and the blockade of the HERG-coded potassium channel by drugs cause the long QT syndrome. In recent years, ten genes have been reported to correlate with LQTS. The mutations in HERG gene channel cause type 2 long QT syndrome(LQT2), which are the second common reason of LQTS. Protein-protein interaction is an important basis for many cellular functions, for exemple,signal transduction, cell cycle regulation, RNA transcription, DNA replication, protein translation, protein post-translational processing and modification functions are dependent on the completion of protein-protein interactions.in recent years,several cellular proteins have been reported to interact with HERG channel and modify its expression, trafficking and function. Fifteen proteins, including PTPN12 (Protein Tyrosine-protein phosphatase non-receptor type 12), caveolin-1;FHL2;Myotrophin,and so on, have been preliminarily screened to interact with the amino terminus of the HERG channel protein by the yeast two-hybrid technique in early research. Among them, PTPN12 may be of great importance to the regulation of HERG channel. So identifying the PTPN12 effects to the expression and function of.HERG channel will lead to reveal novel regulators to HERG channel,, and contribute to the exploitation of antiarrhythmic proteins targeting the HERG channel.Objective:To verify the proteins interacted with HERG potassium channel and study the modulatory effects of the interacting proteins on this channel.Methods:(1) To further confirm the interaction, the Co-immunoprecipitation was performed by using specific antibody.:The mixture of HERG polyclonal antibody and total proteins of the rat myocardium cells was rotated, then to add Protein A/G Plus-Agarose. The sediments were centrifugated from the final mixture for electrophoresis, then for Western blot analysis by PTPN12 antibody. (2) GST pull-down assay. To study the PTPN12-HERG interaction, the GST-HERG-NT fusion protein was expressed and purified, then the proteins pulled down by the GST-HERG-NT was Western blot analyzed using anti-PTPN12 antibody.(3) Immunofluorescence analysis:HEK 293 cells were transiently co-transfected with pcDNA3.0-HERG and pcDNA3.1-PTPN12 using Lipofectamine 2000 (Invitrogen), The co-transfected cells were fixed after 48 h. the anti-PTPN12 antibody and anti-HERG antibody were used to probe the subcellular localization of these two proteins. (4) The patch-clamp technique was used to study the affection of the interacting protein on the HERG channel property.Results:(1) Co-immunoprecipitation. The anti-HERG antibody precipitated the PTPN12 and the HERG complex from the rat heart lysates. (2) GST pull-down assay. The GST-HERG-NT fusion protein, but not the GST protein, pulled the PTPN12 down from the rat heart lysates. (3) After immunofluorescence analysis, we could see HEK293 cells co-transfected with the two plasmids expressing the red fluorescent protein HERG and the green fluorescent proteinPTPN12 in cell membrane with an inverted fluorescent microscope. The two fluorescence overlap (yellow fluorescence) indicated the co-localization of HERG andPTPN12 in co-transfected cells.We detected the co-localization of the PTPN12 and HERG occurred mostly in the membrane surface compartment, where the majority of HERG present. (4) The patch clamp electrophysiological experiment showed that PTPN12 decreases the HERG current amplitude of the HERG potassium channel.Conclusions:Our results show that PTPN12 interacts with the amino terminus of HERG and modulates the HERG potassium channel function in cardiac cell. Through the interaction, PTPN12 decrease the HERG current amplitude. Our findings indicate that PTPN12 involved in the regulation of HERG potassium channels, Its mechanism might be by changing the degree of phosphorylation of the HERG potassium channels. This novel finding may aid in the further understanding the molecular basis of HERG channel diversity and arrhythmogenesis in the long-QT syndrome, also, it may help to contribute to the exploitation of new antiarrhythmic protein drugs targeting the HERG channel.