The Target Of Amantadine Inhibiting M2 Ion Channel And Screening Inhibitors Of BM2

Influenza B virus together with H1N1 and H3N2 of influenza A virus are the major prevalent agents of influenza and bring great loss to human health and economy. Amantadine and rimantadine have been established effectively in the prophylaxis and treatment of influenza A virus infections, but not for BM2 and influenza B virus. It is important to elucidate the mechanism of amantadine inhibiting M2 ion channel, which will be helpful for developing new effective blockers. However there are debates in the binding domain of M2 protein with amantadine. In this report, we constructed chemeric proteins with transmembrane domains of M2 ion channel of influenza A and B virus(AM2 and BM2 respectively), tried to figure out the amantadine binding domain. The results showed that T-REx293 cell expressing M2 or chimeric protein was a good model for studying activity of M2 ion channel. Compared with AM2 and BM2, the chimeric proteins could integrate into membrane but with somewhat decrease of conductance. Accumulation of chimeric proteins in cytoplasm resulted from the structure change of transmembrane, which might contribute to decrease of ion channel activity. The data also showed that the N-terminal extracellular domain of BM2 played important role in integration of BM2 into membrane. The chimeric proteins A36/B19 and B6/A25-36/B19 (N-terminal aa1-18 or transmembrane aa7-18 of BM2 substituted by N-terminal aa 1-36 or transmembrane aa 25-36 of AM2 respectively) showed sensitivity to amantadine, that indicated aa 25-36 of AM2 functioned as amantadine binding domain. With the BM2 stable cell, we developed a GFP based assay for screening inhibitors and got two chemicals with inhibitory activity against BM2 ion channel. Taken together, the amantadine binding domain was determined uesing M2 protein stable cell lines, it has directive significance for studying M2 ion channel activity and screening inhibitors.