Regulatory Role Of Ca²⁺-Calmodulin-dependent Protein KinaseⅡ In Store-operated Calcium Entry

Objective: Store-operated Ca²⁺ entry（SOCE）is one of the main forms of intracellular calciuminflux and regulates a wide range of cellular functions,and its function is the underlying mechanism of many diseases.Although the basic molecular mechanisms of SOCE activation;stromal interaction molecue1（STIM1）translocation to plasma membrane to interact with Ca²⁺ selective channel-forming subunit（Orai1）,is well understood,it is not quite clear about the regulatory mechanisms of STIM1 aggregation,redistribution,and efficiency of the interaction.Ca²⁺-calmodulin-dependent protein kinase Ⅱ（Ca MKⅡ）,a paramount regulator of multiple Ca²⁺ handling proteins and ion channels,is able to translate a diverse set of signalling events into downstream effectors.A variety of cardiovascular diseases is accompanied by abnormal intracellular Ca²⁺,calcium overload,and at the same time the increase of Ca MKⅡ expression or activity of the kinase.In Xenopusoocytes,Ca MKⅡactivation has been proposed to potentiate SOCE,however,it is unknownhow Ca MKⅡ regulating SOCE function.Here,we provide evidence for an up-regulatory role of Ca MKⅡin SOCE in HEK293 cells and He La cells,and further research and explore its regulation mechanism.Methods: Intracellular Ca²⁺ concentration([Ca²⁺]i)was measured using the fluorescent probe Fura-2 in HEK293 cells and He La cells.Protein expression levels of p-Ca MKⅡδ,Ca MKⅡδ,STIM1 and Orai1 were measured using Western blot.Ca MKⅡoverexpression plasmids were constructed and expressed in HEK293 and He La cells.Co-localization of STIM1 with Orai1 and STIM1 aggregationwere measured using Immunoflorescence.Co-immunoprecipitation was utilized to find out STIM1-Orai1 interactions.Results: Inhibition of calmodulin withn-（6-aminohexyl）-5-chloro-1-naphthalenesulfonamide hydrochloride（W-7）or Ca MKⅡ with KN-93 abrogatedthe peak and rate of SOCEand the increased co-localization in PM and interaction between STIM1 and Orai1 induced by store depletion with thapsigargin（TG）.The phosphorylation of Ca MKⅡδ on Thr287 due to TG was also abolished by KN-93 but not by KN-92,suggesting an activation ofthe endogenous Ca MKⅡδ during SOCE.Additionally,over-expression of Ca MKⅡ in both cell lines transfected with Ca MKⅡδ gene for 48 h enhanced the TG-induced STIM1 and Orai1co-localization in cell membraneand theirinteraction as wellas SOCE.Finally,pretreatment of cells with KN-93 or W-7 dramatically decreased STIM1 aggregation in cytosol and perimembrane regions,whereas Ca MKⅡδ overexpression increased it due to TG.Conclusion: Thisstudy demonstrates that Ca MKⅡ activation accompanies SOCE initiation and maintenance,to enhance STIM1 aggregation and redistribute on cell surface to interact with Orai1.Our findings unveil a novel mechanism for Ca MKⅡ regulating SOCE in physiological conditions.