Study of phosphorylation levels of GABAA receptor in cell line of neuroblastoma with knocked down malic enzyme 2 (ME2)

Background:Malic enzymes(ME; EC 1.1.1.40) represent a family of oxidative decarboxylases that catalyze the divalent metal ion(Mn2+ or Mg2+)dependent irreversible oxidative decarboxylation of L-malate to yield CO2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD+ or NADP+. In neurons pyruvate produced from malate is a substrate for the neuronal synthesis of γ-aminobutyric acid (GABA), which is a major inhibitory neurotransmitter in the central nervous system (CNS). The GABAA receptor is a ligand-gated ion channel whose function and activity can be regulated by ligand binding or alternatively may be influenced indirectly through the phosphorylation of specific subunits that comprise the GABAA receptor pentamer. Methods:Cell culture of the cell line was obtained from the American Type Culture Collection at Iscove’s Modified Medium. All environments were placed on calf embryo (10% v/v), 100 units of penicillin and 100 mg/ml streptomycyne, and grown at 37C and 5% CO2. Infected cells with shRNA virus were selected with poromycin 1.0 mg/ml, and the ME2 or ACL stack was used for analysis. PAGE and western blot electrophoresis: After the Bradford test, 34 micrograms of each protein sample are mixed and used in a 1:1 sample with a sample buffer. The molecular weight marker is used to determine the protein’s position.Also, the β-actin antibody is used to load control. Findings:Immunoblotting showed a decrease in GABA (A) R phosphorylation in Knocked down cell line in comparison with the control. Conclusion:Epilepsy is characterized by high and uncontrolled activity, or all central nervous system. Animal studies indicate that the seizure is due to the fluctuation of the thalamic lattice neurons (which are inhibitors of gammaamino-butyric acid production neurons), and the thalamus and cortex-thalamus stimulatory neurons.