Functional Characterization Of Kir Channels In Nlaparvata Lugens And Spodoptera Exigua

Inwardly rectifying potassium channel as an important member of potassium ion channel plays the key roles in controlling the resting membrane potential,maintenance of cellular homeostasis,transduction cell excitability and it's also an important target for developing new therapeutic drugs.In this study,we cloned the Kir genes from Spodoptera exigua and Nilaparvata lugens,analyzed the expression pattern,and characterized the function of Kir1 in Spodoptera exigua and Nilaparvata lugens.In present study,we obtained eight Kir channel genes from Nilaparvata lugens and Spodoptera exigua,analyzed the expression patterns of Kir genes in different growth stages and tissues using quantitative PCR technique.Kir1 channel genes from Nilaparvata lugens and Spodoptera exigua were heterologous expressed in HEK293 cells and inward-rectifier characteristic of Kir1 channel was detected using whole-cell patch-clamp technique.Thus we found the blocking effect of flonicamid on Kir1 channel.According to equivalence of thallium ion and potassium ion,a high-throughput screening technique for insect Kir channels was developed to screen the chemicals that act on Kir channel of insects.1.Identification and phylogenetic analysis of Kir channelsWe got 11 cDNA that encode putative Kir channels,in which 3 cDNA from N.lugens and 8 cDNA were from S.exigua.Deduced protein sequences revealed structural motifs typical of inwardly rectifying K+channels,a conserved pore helix,ion selectivity filter flanked by two transmembrane segments(M1 and M2).Each Kir channel subunit shows the highest areas of conservation in the transmembrane domains and selectivity filter;the latter includes the K+signature sequence "TXGYG",A phylogenetic comparision of Kirs from N.lugens and S.exigua with orthologies Kirs of dipteran insects and human revealed that Kirs from these two insects can be divided into three subfamilies(Kir1,Kir2,Kir3).All the insects Kirs except Kir3 were clustered together and divergent from human Kirs.NlKir3 is clustered with human Kir6 and Kir3 from other insects are close to human Kir6.Sequence alignment also revealed that there were four different splicing transcripts for SeKir2A.2.Expression patterns of Kir channel genes in Nilaparvata lugens and Spodoptera exiguaWe studied expression patterns of Kir channel genes in Nilaparvata lugens and Spodoptera exigua using quantitative PCR technique.In Nilaparvata lugens,the expression level of Kir1 was higher in egg and nymph than adult.Kir2 and Kir3 were both high expressed in nymph.All Kir channel genes of Nilaparvata lugens were highly expressed in antenna,head and abdomen,and Kir1 and Kir2 also had a high expression level in wing.In the growth stages of Spodoptera exigua,Kir1 Kir2A and Kir3 A had a high expression level in larva stage,but Kir2B and kir3B were highly expressed in egg.In the tested tissues,Kir1 had a high expression in fat body,and other Kir genes were highly expressed in midgut,hingut and Malpighian tubules.3.The inward-rectifying characterization of Kir channels from N.lugens and S.exiguaWe characterized the functional properties of NlKir1 and SeKirl channels using whole-cell patch-clamp in HEK293 cells transiently transfected with constructs of SeKirl and NlKir1,respectively.Robust inward potassium currents were recorded in HEK293 cells expressing SeKirl or NIKirl.And the addition of Ba2+completely blocked the inward potassium current.4.Block of K+current by flonicamid and paichongdingThe whole-cell patch clamp recordings from NlKir1 or SeKirl transfected HEK293 cells recorded flonicamid inhibit Kir1 current in a concentration dependent manner in 0.01 to 10?M concentrate range.Flonicamid inhibited NlKir1 and SeKirl channel with IC50 value of 0.64 and 0.16?M,respectively.Further more,paichognding was also found to inhibit NlKir1 and SeKirl channels.The IC50 values were 5.32 and 3.37?M,respectively.However,no inhibitory effect was observed for pymetrozine and imidacloprid.5.Screening plat target at Kir channel and based on technology of thallium ion fluorescenceWe developed a high-throughput screening technique for insect Kir channel-thallium flux assay.The method was based on the permeability of Tl+to potassium channel and the combination with fluorescent dye.The fluorescent intensity indicated the opening of Kir channel.In present study,we developed stably transfected cell lines of NlKirl and SeKirl and tested the inhibitory effect of flonicamid and paichognding in these cell lines.The IC50 of flonicamid in NlKir1 and SeKirl channel were 1.96?M and 4.37 ?M,respectively.Paichongding inhibited NlKir1 and SeKirl with IC50 of 173.6?M 13.99?M,respectively.Pymetrozine and imidacloprid have no inhibition effect on Kir channel at high concentration.