Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.
The A/M2 protein of influenza A virus forms a tetrameric proton-selective pH-gated ion channel. The H(37)xxxW(41) motif located in the channel pore is responsible for its gating and proton selectivity. Channel activation most likely involves protonation of the H37 residues, while the conductive state of the channel is characterized by two or three charged His residues in a tetrad. A/M2 channel activity is inhibited by the antiviral drug amantadine. Although a large number of functional amantadine-resistant mutants of A/M2 have been observed in vitro, only a few are observed in highly transmissible viruses in the presence or absence of amantadine. We therefore examined 49 point mutants of the pore-lining residues, representing both natural and nonnatural variants. Their ion selectivity, amantadine sensitivity, specific activity, and pH-dependent conductance were measured in Xenopus oocytes. These measurements showed how variations in the sequence lead to variations in the proton conduction. The results are consistent with a multistep mechanism that allows the protein to fine-tune its pH-rate profile over a wide range of proton concentrations, hypothesized to arise from different protonation states of the H37 tetrad. Mutations that give native-like conductance at low pH as well as minimal leakage current at pH 7.0 were surprisingly rare. Moreover, the results are consistent with a location of the amantadine-binding site inside the channel pore. These findings have helped to define the set of functionally fit mutants that should be targeted when considering the design of novel drugs that inhibit amantadine-resistant strains of influenza A virus.
Balannik
Victoria
V
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Carnevale
Vincenzo
V
Fiorin
Giacomo
G
Levine
Benjamin G
BG
Lamb
Robert A
RA
Klein
Michael L
ML
Degrado
William F
WF
Pinto
Lawrence H
LH
D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D024882:Drug Resistance, Viral; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D013329:Structure-Activity Relationship; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000086082:Viroporin Proteins
D000998:Antiviral Agents; D007473:Ion Channels; D011522:Protons; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins