Oxygen And Glucose Deprivation Related Protein Annexin A6 And Potassium Channel Protein KCNQ2 Interaction Research

Stroke is a cerebral blood circulation disorder diseas e caused by an abrupt blockage of arteries,and the blood supply to the brain is blocked,lack of oxygen and glucose depletes the cellular energy and hence leads to a wide range of deleterious cellular reactions.The current effective drug treatment is mainly limited to the dissolution of thrombus by tPA to alleviate the disease.Therefore,the mechanism behind the stroke which causes neuron damages is needed to be explored and which would be of great interest for the clinical treatment and the development of new drug.In our study we focused on proteins which may play role s in neuronal damage during ischemic stroke,the primary cultured neuron was deprived oxygen and glucose to imitate ischemia stroke in vitro.Extracting neuronal total protein,p olyacrylamide gel electrophoresis,coomassie blue staining,select the significant difference protein band contrast to control and identify Annexin VI by mass Spectrometry.Annexin VI belongs to a family of calcium-dependent membrane and phospholipid binding proteins,involved in a series of physiological process like plasma membrane repair,apoptotic signaling pathway,calcium ion binding,regulation of muscle contraction.Immunofluorescence and immunoblotting result shows that Annexin VI was downregulated in the neurons with the deprived condition of oxygen and glucose.We confirmed the predicted interaction between the Annexin VI and KCNQ2 with the help of co-immunoprecepitation,while chelating Ca²⁺significantly decline the interaction.We also conducted a series of coIPs in HEK293 cells that expressed eGFP-tagged WT or other truncated forms of Annexin A6 to identify regions of Annexin A6 that mediate its binding to KCNQ2,indicating that deletion Annexin A6N-terminal core region significantly reduce the interaction.KCNQ2 is potassium channel protein that plays an important role in maintaining electrical excitation of neurons and transmission of synapses.The arginine mutations at positions 207 and213 of KCNQ2 cause an abnormality in ion channel function,and th e two sites are highly conserved.In order to explore the protein interaction when KCNQ2 is site,.213 mutation does not affect protein interaction,and when 213 arginine is mutated to glutamine,the interaction is attenuated.Collectively,this study reveals that OGD stimulation leds to down-regulation of Annexin A6 expression,and co-immunoprecipitation identified the interaction between Annexin A6 and KCNQ2.Further studies indicate that Annexin A6N-terminal core region plays an important role in the protein interaction,in addition,KCNQ2 213 arginine mutation affect the protein interaction.The above results provide the basis for subsequent cellular damage mechanisms.