Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel

Xianghong Jing, Chunlong Ma, Yuki Ohigashi, Fernando A. Oliveira, Theodore S. Jardetzky, Lawrence H. Pinto, Robert A. Lamb

Research output: Contribution to journalArticlepeer-review

134 Scopus citations

Abstract

Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596-599] and by NMR methods [Schnell and Chou (2008) Nature 451:591-595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24-36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.

Original languageEnglish (US)
Pages (from-to)10967-10972
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number31
DOIs
StatePublished - Aug 5 2008

Keywords

  • Amantadine inhibition of influenza virus
  • Amantadine resistance
  • Drug-binding site
  • Influenza reverse genetics

ASJC Scopus subject areas

  • General

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