1.A.19 The Type A Influenza Virus Matrix-2 Channel (M2-C) Family

The M1 and M2 matrix proteins of influenza virus type A are produced by alternative splicing of RNA segment 7, but only the first 9 residues are shared by the two proteins. The M2 'matrix' protein is a 97 amino acyl residue long integral membrane protein with an N-terminal extracellular domain (residues 1-23), a single transmembrane domain (residues 24-44) [D₂₄PLVVAASIIGILHLILWILD₄₄] and a large cytoplasmic domain (residues 45-97). It associates into pseudosymmetric homotetramers of parallel transmembrane α-helices with a tilt angle of 25-32&730; (Tian et al., 2002). These four hydrophobic α-helices form the proton-selective channel. A 25-residue synthetic peptide of the same sequence forms a H⁺-selective channel with properties similar to those of the native 97-residue protein. Detailed structural information regarding the transmembrane domain of M2 is available. A two tetramer structure has been solved at 2.1 Å resolution with and without the inhibitor, amantadine. M2 resembles the NB glycopeptide ion channel of influenza virus type B (TC #1.A.32) in size, topology and function but lacks sequence similarity with it.

The M2 channel protein is an essential component of the viral envelope because of its ability to form a highly selective, pH-regulated, proton-conducting channel. The virus enters the cell by internalization via the endocytic pathway. Viral uncoating, facilitated by the M2 H⁺ channel, takes place in the endosomes. The M2 channel allows protons to enter the virus’ interior, and acidification weakens the interaction of the M1 protein with the ribonuclear core. M2 also modulates the pH of the trans-Golgi network. The anti-influenza virus drug, amantadine, is a specific blocker of the M2 H⁺ channel. In the presence of amantadine, viral uncoating is incomplete, and the ribonucleoprotein core fails to promote infection. M2 exhibits 10⁷ x selectivity for H⁺ over K⁺ (Moffat et al., 2008).

Mechanistic analyses of the M2 channel have provided evidence against a mechanism involving the flux of hydronium ions (H₃O⁺) through a channel. Instead, H⁺ is believed to interact with titratable histidyl groups within the channel. In this mechanism, two histidines in two separate subunits exist, one protonated (His⁺), the other unprotonated (His⁰), and the channel is open to the outside. When His⁰ binds H⁺, electrostatic repulsion causes a conformational change in the channel so that it opens inwardly. When the H⁺ passes in, the conformation reverts. This 'carrier-type' mechanism is dependent on the lipid environment and is consistent with the slow rate observed for H⁺ transport (Cady et al., 2007; Duong-Ly et al., 2005). The channel is also capable of transporting NH₄⁺ and other cations.

The indole moiety of the single transmembrane tryptophan residue (position 41) is responsible for H⁺ gating (Tang et al., 2002). Thus, the side chain of Trp⁴¹ probably blocks the pore when the pH_out is high so it is closed. When the pH is low, this side chain leaves the pore so it is open.The determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide; residues 22-46 in M2 of 97aas (Ma et al., 2009).

Stouffer et al. (2008) described the crystal structure of the transmembrane-spanning region of the homotetrameric M2 protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. A multistep mechanism allows the protein to fine-tune its pH-rate profile over a wide range of proton concentrations, arising from different protonation states of the H37 tetrad (Balannik et al., 2010).

In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. The structure of the tetrameric M2 channel in complex with rimantadine has also been determined by NMR (Schnell and Chou, 2008). In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.  Amantadine binds to the pore (Jing et al., 2008).

The generalized transport reaction catalyzed by the M2 channel is:

H⁺ (out) H⁺ (in).