Identification Of Cardiac Connexin43 Carboxylic Terminal Interacting Protein Using The Yeast Two-hybrid System

Background: Gap junctions(GJs) are specialized channels by which the electrical and chemical information communicate between the neighboring cardiomyocytes, and the connexin43 (Cx43) is the predominant protein consisting the gap junction localized in ventricle. The gap junctional intracellular communication(GJIC) plays a key role for the coordinated contraction and synchronizing coupling in the global heart. In recent years, a growing body of evidence showing that the abnormality of the cardiac electrical coupling mediated by GJ, as another vital factor, contributes more to the arrhythmogenesis.than the abnormality of ion channel does. And GJs are also crucial regulators for heart gene transcription, development and protection of ischemic cardiomyocytes due to the second messengers communication.The function of the cardiac GJ is regulated by different factors including intracellular PH, concentration of intracellular Ca²⁺ and ATP, the phosphorylation status of the Cxs, the voltage difference between two end of the channel, and some neurohumoral factors, etc. And most of the regulating sites locate at the Cx43 cytoplasmic carboxylic terminal. More recently ,we knew that Protein-Protein interaction dominates the cell function. So identifying the Cx43 binding proteins in human heart, will lead to understanding the underlying mechanism of GJ channel protein RNA transcription,protein translation,post-translational processing,protein targeting and localization,quantity control and protein degradation. Furthermore the binding proteins identification will also help to reveal the novel GJ channel regulators, and ultimately this finding will contribute to the exploitation of antiarrhythmic proteins targeting the GJ channel. AIM: Our goal in this research is to identify the Cx43 binding proteins in human heart by conducting a yeast 2-hybrid screen.METHODS:â‘ A cDNA cassette encoding the cardiac gap junctional connexin43 carboxylic terminal (AA235-382) introduced with EcoRI and Bali restriction enzyme cutting sites was PCR amplified, pGBKT7-Cx43-CT was constructed by digesting the PCR product with EcoRI and BamHI restriction enzymes followed by ligation to EcoRI/BamHI digested pGBKT7 plasmid using T4 DNA ligase;â‘¡The pGBKT7-Cx43-CT was transformed into AH109 yeast cells using the chemical method;â‘¢The extraction of protein from the transformed AH109 yeast cells was accomplished by the "Urea/SDS Method";â‘£Detect the expression of "bait" protein (Gal4-BD-C-myc-Cx43-CT fusion protein) by performing the Western blot using anti-c-myc antibody;⑤Determine whether the bait itself auto-activated the reporter genes, and whether it is necessary to inhibit the leaky His3 expression by using the 3-AT with the optimized concentration;â‘¥The AH109 transformed with the pGBKT7-Cx43-CT mated with the human heart cDNA library, and the probable protein interaction were retest in yeast, then the positive clones were sequenced;⑦The sequencing results were analyzed for homology in NCBI BLAST database.RESULTS:â‘ The "bait" plasmid was verified by sequencing, ensuring that the Cx43-CT was inframed with the "Gal4 DNA binding domain-C-myc" and the PCR mediated mutations were not inadvertently introduced;â‘¡The "bait" plasmid was transformed into AH109 yeast cells with 100 percent success rate;â‘¢Total protein with approving concentration was extracted from transformed AH109 yeast cells;â‘£The western blot has confirmed the stable expression of the Gal4-BD-C-myc-Cx43-CT fusion protein;⑤There was no auto-activation by the "bait" protein, but weak leaky His3 expression was found, which could be inhibited by 5 mM/L 3-AT;â‘¥After mating with a human Heart cDNA library and retested in yeast, 10 tentative positive colonies were obtained, seven "prey" proteins were confirmed.CONCLUSION" Multiple proteins in cardiac myocyte probably interact with the cardiac gap junctional connexin43 carboxylic terminal, furthermore regulate the GJ channel and involved in diverse pathophysiologic processes.