Phosphorylation Of Connexin 43 By Cdk5 Modulates Neuronal Migration During Embryonic Development

Objective:Gap junction intercellular communication(GJIC)is a way of information exchange for cell to cell by gap junction as a structural basis and is closely related to some biological functions such as the development of the embryo,cell proliferation and differentiation,homeostasis and so on.Gap junctions(GJ)are made.up of two hemichannels(also known as connexons);each hemichannel is composed of six connexin(Cx)proteins.Two hemichannels distributed in the cell membrane of different type or the same type of cells form a gap junction,which makes the adjacent cytoplasm directly interlinked and provides a direct access for the intercellular transfer of matter and information.The gap junction channels are widely distributed in the central nervous system and make all kinds of cells to a cohesive group by electrical coupling,which play an important role in the regulation of physiological function such as cell growth,differentiation.Connexin43(Cx43),the predominant gap junction protein,is chosen to abundantly express and present in astrocytes in the mature brain tissues.It makes the reciprocal communication between astrocytes and forms a powerful syncytium to play an important role.Neurons are seen as the main functions of the cells in the central nervous system,however,the expression of Cx43 protein in neurons and its function remains unclear.Here we tried to analyze the expression and function of Cx43 in neurons,identity a relationship involving Cx43 and cyclin-dependent kinase 5(Cdk5),discuss the molecular mechanism of Cdk5 regulation of Cx43.Methods:(1)The expression of Cx43 in rat cortex neurons was tested by RT-PCR,western blotting and immunofluorescence staining;(2)The neural cells specific Cx43 knockout mice were constructed by Cre/loxP recombination system and the migration of neural cells in the developing cerebral cortex between wild type and knockout mice was analyzed by immunofluorescence staining;(3)The behavior changes of neuron-specific Cx43 conditional knockout mice were detected by using a range of behavioral tests;(4)The expression level of Cx43 protein and Cdk5 protein in brain tissue of different periods of embryo development was analyzed by western blotting;(5)The Cdk5-related phosphorylation site of Cx43 was predicted with bioinformatical tools(GPS2.1,Scansite3);(6)Co-immunoprecipitation(Co-IP)demonstrated the interaction between Cdk5 and Cx43;(7)Application of vitro kinase experiment(in vitro kinase assay)to validate Cx43 phosphorylation site;(8)The wild type plasmid or mutant Cx43 plasmid of phosphorylation sites were transfected to HeLa cells and phosphorylation statuses were analyzed by specific phosphorylation antibody;(9)The wild type plasmid or phosphomimetic mutant plasmid were transfected to HeLa cells and the expression levels of Cx43 protein in cell membrane were analyzed;(10)The degradation levels and pathways of Cx43 protein were tested by western blotting.Results:(1)Cx43 is expressed in neurons in the early development stage of brain;(2)Conditional knockout of Cx43 in neurons impairs neuronal migration in embryonic development;(3)Neuron-specific Cx43 conditional knockout mice exhibit normal learning and memory functions at the adult stage;(4)Neuron-specific Cx43 conditional knockout mice exhibit altered anxiety-related behavior at the adult stage;(5)The expression of Cx43 in neurons is negatively regulated by Cdk5;(6)Cx43 is a phosphorylation substrate of Cdk5;(7)Phosphorylation of Cx43 by Cdk5 on Ser279 and Ser282 influences the membrane expression of Cx43;(8)Phosphorylation of Cx43 by Cdk5 on Ser279 and Ser282 promotes the proteasome-dependent degradation of Cx43.Conclusion:During embryonic development,the neuronal expression of Cx43 plays an important role in neuronal migration;Neuron-specific Cx43 conditional knockout mice exhibit altered anxiety-related behavior at the adult stage;Along with the development,Cdk5 directly phosphorylates Cx43 at Ser279 and Ser282 inhibits its membrane targeting and promotes its degradation.Thus,the neuronal expression of Cx43 reduced and Cdk5 phosphorylation downregulated Cx43 in mature neurons.