Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel
Xianghong Jing1, Chunlong Ma, Yuki Ohigashi
1Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA.
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.
Insights
Influenza A/M2 and B/BM2 ion channels are targeted by amantadine and rimantadine. Functional studies suggest drug binding outside the pore, not within, is key for inhibiting influenza A/M2 channel activity.
Area of Science:
- Biophysics
- Virology
- Structural Biology
Background:
- Influenza A and B viruses possess proton-selective ion channels, A/M2 and BM2, respectively.
- Amantadine and rimantadine are drugs that inhibit A/M2 channel activity.
- Previous structural studies (X-ray crystallography and NMR) proposed different drug-binding sites for amantadine and rimantadine within or outside the A/M2 channel pore.
Purpose of the Study:
- To investigate the precise binding site of amantadine and rimantadine on the influenza A/M2 ion channel.
- To elucidate the mechanism of pharmacological inhibition of the A/M2 channel by these antiviral drugs.
- To determine if drug binding outside the pore is the primary mechanism for A/M2 channel inhibition.
Main Methods:
- Site-directed mutagenesis of key residues (D44 and R45) in the A/M2 channel.
- Electrophysiological recordings in Xenopus laevis oocytes and mammalian cells to assess channel activity and drug sensitivity.
- Introduction of mutations into the influenza virus genome.
- Construction of a chimeric BM2 channel by transplanting A/M2 pore residues.
Main Results:
- Mutagenesis of D44 and R45 did not render the A/M2 channel insensitive to amantadine, contradicting models of binding within the pore.
- Mutations D44A and R45A, and their introduction into the viral genome, still allowed for amantadine inhibition.
- A chimeric BM2 channel incorporating A/M2 pore residues showed partial amantadine inhibition, suggesting pore interactions are not solely responsible for inhibition.
Conclusions:
- The primary site of amantadine and rimantadine binding responsible for pharmacological inhibition of the influenza A/M2 ion channel is likely located outside the channel pore.
- Existing models suggesting direct pore occlusion by amantadine or rimantadine may not fully explain the observed inhibition.
- Further research is needed to fully characterize the extracytoplasmic binding site and its role in antiviral drug efficacy.
More Related Videos
08:31Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions
Published on: December 1, 2020
09:07Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses
Published on: January 20, 2017
Related Concept Videos
01:25Inhibitors Of Virion Release
01:30Ligand-Gated Ion Channel Receptor: Gating Mechanism
01:15Antifungal Agents
01:27Influenza
01:19Ligand-gated Ion Channels
Three Subfamilies of Ligand-gated Ion Channels
Ligand-gated ion channels fall into three subfamilies. The 'Cys-loop' includes the nicotinic acetylcholine receptors, γ-aminobutyric acid (GABA), glycine, and 5-hydroxytryptamine receptors. The second one is the 'Pore-loop' channels that include the...
01:21Drugs Affecting Neurotransmitter Release or Uptake