title: A dimer is the minimal proton-conducting unit of the influenza a virus M2 channel.
abstract: When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachicho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: kawano.kenichi.2u@pharm.kyoto-u.ac.jp.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kawano
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachicho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: yyano@pharm.kyoto-u.ac.jp.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Yano
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachicho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: mkatsumi@pharm.kyoto-u.ac.jp.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Matsuzaki
mesh_terms: D000547:Amantadine / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016466:CHO Cells; D002784:Cholesterol / Q000737:chemistry; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D055503:Protein Multimerization; D011522:Protons; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D000547:Amantadine; D000998:Antiviral Agents; D002784:Cholesterol; D006639:Histidine; D011522:Protons; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins
keywords: FRET; M2 proton channel; coiled-coil labeling; dimer–tetramer equilibrium; spectral imaging