The M2 proton channel of influenza A virus is a tetrameric integral membrane protein that forms pH-gated proton channels in the viral lipid envelope. The low pH of endosomes activates the M2 channel before hemagglutinin-mediated fusion, acidifying the viral interior and facilitating the dissociation of the matrix protein from viral nucleoproteins, a necessary step for viral genome release. In the trans-Golgi network (TGN), M2 prevents premature conformational changes in hemagglutinin by equilibrating TGN pH with the host cell cytoplasm. Inhibiting M2 proton conductance with drugs like amantadine or rimantadine inhibits viral replication. The structure of the M2 channel in complex with rimantadine was determined by NMR, revealing a closed conformation with a narrow channel defined by four tightly packed transmembrane (TM) helices. A "tryptophan gate" is locked by intermolecular interactions with aspartic acid, while a C-terminal amphipathic (AP) helix forms an inward-facing base. Lowering the pH destabilizes the TM helical packing, unlocking the gate and allowing proton conduction. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel, stabilizing the closed conformation. Drug-resistance mutations are predicted to counteract drug binding by increasing pore hydrophilicity or weakening helix-helix packing, facilitating channel opening. The M2 protein is a 97-residue single-pass membrane protein with its N- and C-termini directed toward the outside and inside of the virion, respectively. The four TM helices form a channel with His37 as the pH sensor and Trp41 as the gate. Adamantane-based drugs like amantadine and rimantadine target the M2 channel and have been used as antiviral drugs against influenza A viruses. Structural models of the M2 channel have been built based on sequence analysis, mutagenesis, and solid-state NMR. However, many studies have been done on inherently unstable TM-only constructs, leading to conflicting conclusions. The M2(18–60) construct, including 15 residues of the C-terminus, forms a stable tetramer in DHPC detergent micelles and yields high-resolution NMR spectra. In the closed conformation at pH 7.5, M2(18–60) is a homotetramer with an unstructured N-terminus, a channel-forming TM helix, a short flexible loop, and a C-terminal AP helix. The TM helices assemble into a four-helix bundle with a left-handed twist angle of ~23° and a well-defined pore. His37 and Trp41 are inside the pore, with His37 in a trans rotamerThe M2 proton channel of influenza A virus is a tetrameric integral membrane protein that forms pH-gated proton channels in the viral lipid envelope. The low pH of endosomes activates the M2 channel before hemagglutinin-mediated fusion, acidifying the viral interior and facilitating the dissociation of the matrix protein from viral nucleoproteins, a necessary step for viral genome release. In the trans-Golgi network (TGN), M2 prevents premature conformational changes in hemagglutinin by equilibrating TGN pH with the host cell cytoplasm. Inhibiting M2 proton conductance with drugs like amantadine or rimantadine inhibits viral replication. The structure of the M2 channel in complex with rimantadine was determined by NMR, revealing a closed conformation with a narrow channel defined by four tightly packed transmembrane (TM) helices. A "tryptophan gate" is locked by intermolecular interactions with aspartic acid, while a C-terminal amphipathic (AP) helix forms an inward-facing base. Lowering the pH destabilizes the TM helical packing, unlocking the gate and allowing proton conduction. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel, stabilizing the closed conformation. Drug-resistance mutations are predicted to counteract drug binding by increasing pore hydrophilicity or weakening helix-helix packing, facilitating channel opening. The M2 protein is a 97-residue single-pass membrane protein with its N- and C-termini directed toward the outside and inside of the virion, respectively. The four TM helices form a channel with His37 as the pH sensor and Trp41 as the gate. Adamantane-based drugs like amantadine and rimantadine target the M2 channel and have been used as antiviral drugs against influenza A viruses. Structural models of the M2 channel have been built based on sequence analysis, mutagenesis, and solid-state NMR. However, many studies have been done on inherently unstable TM-only constructs, leading to conflicting conclusions. The M2(18–60) construct, including 15 residues of the C-terminus, forms a stable tetramer in DHPC detergent micelles and yields high-resolution NMR spectra. In the closed conformation at pH 7.5, M2(18–60) is a homotetramer with an unstructured N-terminus, a channel-forming TM helix, a short flexible loop, and a C-terminal AP helix. The TM helices assemble into a four-helix bundle with a left-handed twist angle of ~23° and a well-defined pore. His37 and Trp41 are inside the pore, with His37 in a trans rotamer