Interaction Between Protein KCNA5 And Protein FHL2

Background: KCNA5 channel (Kv1.5 potassium channel) encoded by KCNA5 (Potassium voltage-gated channel, shaker-related subfamily, member 5) gene mediates an atrial-specific, ultra-rapid delayed rectifier K+ current (Ikur). Ikur only exists in atrium. The development and progression of atrial fibrillation (AF) closely associate with atrial Ikur dysfunction, thus KCNA5 channel is recognized as a promising target for AF treatment. FHL2 consists of four and a half LIM domains, which belongs to LIM-only protein family, and FHL2 has been well studied as a member of FHL family. FHL2 plays role on multiple signals transcription and diseases development and progression via interaction with transcription factors, signal transducers and transduction cofactors. FHL1, another member of FHL family ( identical with FHL2 of 47.9% amino acid), interacts with protein KCNA5 and regulates protein KCNA5 expression and function. FHL1 mainly expresses in skeletal muscles while FHL2 expresses in cardiomoycytes. Therefore, elucidation of the relationship between protein KCNA5 and FHL2 will be beneficial for profoundly understanding the regulation mechanism of cardiac protein KCNA5 and also providing theoretical evidences for developing ideal medicines for AF treatment.Objective: To primarily confirm the interaction between protein KCNA5 and FHL2 by co-immunoprecipitation assays and immunofluorescence analysis.To provide evidences for further study of regulation of protein KCNA5 expression.Methods: (1) Plasmid construction: cDNA encoding KCNA5 channel was used as template and the two terminals of the open reading frame were added Hindâ…¢(5'terminal)and EcoRâ… (3'terminal)two restriction sites and removed termination codon by PCR-amplified. Then the pcDNA3.1/myc-His C plasmid and PCR production were cut by EcoR I/Hindâ…¢respectively. The linear pcDNA3.1/myc-His C and PCR production were connected by DNA linkage enzyme. It was finished that the construction of pcDNA3.1/myc-His C-KCNA5 plasmid. The pcDNA3.0-FHL2 plasmid was provided by my Master Instructor (identified by sequencing already). (2) Cell culture and gene transfection: HEK293 cells were cultured and co-transfected with pcDNA3.1/myc-His C-KCNA5 plasmid and pcDNA3.0-FHL2 plasmid by Lipofectamine 2000. (3) Co-immunoprecipitation: The mixture of FHL2 polyclonal antibody (Rabbit IgG) and total proteins of the co-transfected 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 myc-tag antibody. (4) Immunofluorescence cytochemistry assay: The plasmids pcDNA3.1/myc-His C-KCNA5 and pcDNA3.0-FHL2 co-transfected into HEK293 cells. The co-transfected cells were fixed after 48 h. Protein FHL2 was detected by FHL2 polyclonal antibody (goat IgG) and Alexa-488 donkey anti-goat secondary antibody. Meanwhile, KCNA5 channel was detected by myc-tag rabbit polyclonal antibody and Alexa-555 donkey anti-rabbit secondary antibody.Results: (1) The plasmid pcDNA3.1/myc-His C-KCNA5 was verified by sequencing, ensuring that the KCNA5 was inframed with the myc-His and mutations were not inadvertently introduced. (2) Proteins were extracted from HEK293 cells, which were co-transfected with pcDNA3.1/myc-His C-KCNA5 and pcDNA3.0-FHL2, and proteins were used to co-immunoprecipitation and western blot. An approximately 75Kd protein band which were accordant with KCNA5-myc-His fusion protein were detected in both experimental group and positive group. (3) Co-transfected HEK293 cells staining by immunofluorescence were observed under inverst-immunofluorescent microscopy. Red conjugated protein KCNA5 and green conjugated FHL2 were both observed on cellular membrane. Yellow which overlap with red and green indicated co-localization of these two proteins.Conclusions:Protein KCNA5 interacting FHL2 was identified by co-immuoprecipitation assay and cellular immunofluorescence, and it was beneficial to figure out the regulatory mechanism of KCNA5 channel (Kv1.5 potassium channel) and profoundly understand the pathogenesis of AF.