Intracellular Potassium Homeostasis Involved In The Mechanism Of Neuronal Apoptosis

The decrease of intracellular potassium concentration ([K+]i) is one of the features of cell apoptosis. Recent studies demonstrated that [K+]i decrease is not only a consequence of apoptosis, but also the permission for apoptotic progression. Preventing potassium efflux with potassium channel blockers, or high extracellular potassium concentration, protect various cell types from death induced by different insults. The importance of low [K+]i to apoptotic process has been convincingly proved in cortical neurons and lymphocytes. However, two questions remain opened: 1, which pathway induces the intracellular potassium loss? 2, how low [K+]i promotes apoptotic process? It has been reported that the new protein synthesis is required for the neuronal death related to intracellular potassium loss. Therefore, we investigated whether the alteration of [K+]i regulates transcription of apoptotic proteins, in order to discover the signal transduction pathway that intracellular potassium loss promotes apoptosis. Serum deprivation causes cortical neuronal death through elevating the potassium outward current in early phase of cell apoptosis. Blockade of the potassium efflux with tetraethylammonium (TEA), a global potassium channel blocker, prevents cortical neuronal death independently of cellular depolarization or calcium influx. We established this neuronal apoptotic cell model related to intracellular potassium loss in our laboratory, and examined whether the activity of NF-κB, a transcription factor that has been reported involved in neuronal death, was regulated by alteration of [K~+]i. By using electrophoretic mobility shift assays (EMSAs), Western blotting, Immunocytochemistry and Chromatin immunoprecipitation (ChIP), we demonstrated that serum deprivation caused the degradation of IκBα(the cytosol inhibitory protein of NF-κB), the possession of DNA-binding ability and the nuclear translocation of NF-κB, and the actually binding to endogenous loci within NF-κB downstream genes in vivo. Preventing intracellular potassium loss by TEA did not affect NF-κB activation and DNA-binding in intact cells induced by serum deprivation, suggesting that those processes were independent of alteration of [K+]i. However, preventing [K+]i change did suppress the binding of NF-κB to endogenous loci in vivo and the transcription efficiency of NF-κB target genes, Bcl-XS, IκBαand κB-luciferase. EMSAs in vitro proved that the alteration of potassium concentration affected directly NF-κB/DNA interaction without effect on interaction between the subunits which composed the NF-κB complex. Moreover, the direct action of potassium on the transcription factors/DNA interaction has selectivity, because the DNA binding activity of other neuronal transcription factors, Sp1, E2F and STAT were not sensitive to potassium concentration change, although Forkhead transcription factor exhibited the same K+ sensitivity as NF-κB. Taken together, we concluded that the low potassium concentration in neurons induced by serum deprivation helps the binding of NF-κB to its target genes'promoter regions and improves the transcription. We further examined the roles of NF-κB and Bcl-XS in neuronal death by using RNA interference. We found that down-regulation of p65, the major subunit of NF-κB in cortical neurons, or Bcl-X protein levels survived neurons from serum-deprived treatment. ChIP assays and Western blotting demonstrated that the expression of Bcl-XS was indeed the consequence of NF-κB activation in mature neurons. Thus, our experiments supported that the activation of NF-κB/Bcl-XS pathway promotes the apoptosis of mature neurons. Our study provided the signal transduction pathway how low [K+]i participates in neuronal apoptosis and emphasized the possibility that intracellular ionic strength is involved in regulation of intracellular signal transduction. Furthermore, we proved that NF-κB, which role is controversial in nervous system, is essential to pro-apoptosis in mature neurons through transcription of the pro-apoptotic protein,Bcl-XS.The electrical activity of neural circuit is important to neural plasticity or neuronal survival in central nervous system. The property of voltage-gated potassium (Kv) channels is essential to neuronal excitability and modulates the electrical activity of neural circuit. The modification of Kv proteins may regulate transmitter release from pre-synapse through alteration of the potassium current, or regulate synaptic transmission efficiency through affecting the EPSC integration in post-synaptic neurons. The modification of Kv proteins also may activate other signal transduction pathway to affect the functions of neurons involved in neural circuit. Furthermore, the long-term modification of Kv proteins may change the electrical property of channels, resulting in low intracellular potassium concentration that promotes neuronal apoptosis while neurons are repeated firing in neural circuits. The general modification on Kv proteins mediated by neural circuitry activity is Serine/Threonine phosphorylation. We downloaded the sequences of all the mammalian Kv proteins from NCBI and performed the alignment through software Genedoc. The alignments presented a potent PKA phosphorylation site located in highly conserved T1 domain in all the Kv1 proteins. Using a synthetic peptide with the same sequence as T1 domain that contains the potent PKA phosphorylation site as substrate, we found that PKA indeed phosphorylated this site in vitro. For investigating whether there are endogenous Kv1 proteins phosphorylated on this site in neurons, we raised an antibody, 1505-pV, which specifically recognized the phosphor in the potent PKA phosphorylation site within T1 domain by an antigenic phosphorylated peptide 1505-p. The phosphorylation-specific antibody 1505-pV...