Calcium Release-activated Calcium Channel Modulator 1 Participates In The Steroid Hormone 20-hydroxyecdysone-induced Calcium Influx

Research background, scientific questions and research significanceThe calcium (Ca2+) is a very important ion regulating many physiological activities, including cell division, cell growth, cell progress death, tissue differentiation and the development of the tissue. The Ca2+ has a low water-solubility and the concentration of the Ca2+ in the cells is under strict control as about 100 nM, in contrast, the concentration of the extracellular Ca2+ is about 1 mM. After the simulation, Ca2+ is released from the endoplasmic reticulum (ER) or conducted into cells, Ca2+ combines with Calmodulin and then activates the Calmodulin kinase (CAMK) and regulates the downstream genes. Finally, the Ca2+ is conducted into the ER or outside of the cells through the calcium pumps. There are variety of Ca2+ channels to regulate the cell Ca2+ concentration. The SOCE (store-operated calcium entry) is an important regulatory mechanism, it is the process begins after the stimulation, the IP3 receptor on ER deplets the ER Ca2+ store and sensed by stromal interaction molecule 1 (Stiml), then the calcium release-activated calcium channel modulator 1 (Orail/CRACM1) on the PM is activated and conducts the Ca2+ into cells and restore in the ER. The Ca2+ is also very important in the insects. The development of insects includes metamorphic molting stages. During the metamorphic stages, the concentration of the Ca2+ is regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH). In our former studies, Ca2+ influx was detected under 20E simulation, but the mechnism was unclear. Our study reveals the 20E-induced Ca2+ influx channels, whether Orail is involved in the pathway, the regulating mechanism of Orai1 and the functions in the metamorphic molting stages. Our study supplements the 20E-induced Ca2+ influx and the mechanism of the metamorphic molting; it also provides target genes to control the pests in application.ResultsThe Lepidoptera insect Helicoverpa armigera is a holometabolic insect. We used the insect and the HaEpi to study the function of Orai1 and the 20E-induced Ca2+ influx through the hormone treatment, RNA inference and overexpress in the cells. Here are the results:Orail highly expressed during the metamorphic molting stages in the insects,20E upregulated the express of Orail in the insect and cells; the knockdown of Orail in the insects delayed the metamorphic molting and pupation; knockdown of Orai1 could also repress the expression of the 20E-downstream genes. These results show that Orai1 is regulated by 20E and involved in the development and metamorphic molting. The 20E-induced Ca2+ influx had two peaks, the inhibitor of IP3 receptors XeC blocked both of the peaks; the knockdown of the Orai1 could block the second peak. These results show that 20E induces the SOCE conducted by Orail.In the HaEpi, overexpression of Orail or Stiml seperately did not influence the peaks of the influx; whereas, overexpression of both Orail and Stiml increased the second peak obviously. These results show that Orail functions with Stiml to conduct the Ca2+ influx.Overexpression of Orail-GFP and Orail-RFP in the HaEpi cells, Co-IP showed an interaction of them; 20E treatment could increase the interaction obviously, the increase was block by the inhibitor of GPCR and PLC, the knockdown of ErGPCR1、PLCG1 or Stim1 blocked the increase as well. These results show that Orai1 exists as dimers,20E promotes Orai1 aggregation and activates the Orail.ConclusionThese results show that:the 20E-induced Ca2+ influx is SOCE conducted by Orai1, which is activated through aggregation.Innovation and importance of the researchOur study reveals the SOCE in the 20E-induced Ca2+ influx and the regulating mechanism, and the functions of Orai1 in the metamorphic molting stages; it also provides target genes to control the pests in application.