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

The mechanisms and functions of viral channel proteins have been reviewed by Fischer and Hsu (2011) and Fischer et al. (2012). 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 25-32o; (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 (Pielak and Chou, 2010). Not only the character of the membrane environment, but also the abundance of the phospholipid environment is important to achieve the M2 native structure (Wright et al. 2022).  Large-scale reorientations of transmembrane helices during Influenza A M2 channel opening have been described (Paschke et al. 2023).  The close state of influenza A M2 transmembrane domain is responsive to subtle structural changes of adamantyl amines (Georgiou et al. 2023).

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.  Aminoadamantanes, including amantadine and rimantadine have been widely abandoned due to virus resistance, but thermodynamic considerations allow design of new derivatives (Homeyer et al. 2015).  M2 exhibits 107 x selectivity for H+ over K+ (Moffat et al., 2008). ~250 H+ are transported per second, and the pKm is about 4.7 (Ivanovic et al., 2012).

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 M2 amphipathic helices facilitate pH-dependent conformational transition in influenza A virus (Torabifard et al. 2020).

Acharya et al., (2010) suggested that the conduction mechanism involves the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2(+) and 3(+) with a pKa near 6. A 1.65 A resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms near by. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models (Acharya et al., 2010).

Leiding et al. (2010) showed that M2 has antiporter-like activity, facilitating K+ or Na+ efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K+ to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.

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 pHout 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 M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation (Wang et al., 2012).

The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Alanine 18 of Vpu has been mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. The mutated Vpu TMS resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TMS. Wang et al. (2013) found the reverse when histidine 37 of the HXXXW motif in M2 was mutated to alanine; it decreased the helix tilt by 10° from that of wild-type M2. The tilt change was independent of both the helix length and the presence of tryptophan. Compared to wild-type M2, the H37A mutant displayed a lowered sensitivity to the proton concentration. The solvent accessibility of histidine-containing M2 was greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrate the significance of histidine in a transmembrane helix and the plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.

Water molecules within the pore form hydrogen-bonded networks or 'water wires' from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium ions via second-shell interactions involving bridging water molecules.  A collective switch of hydrogen bond orientations may contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle (Thomaston et al. 2015). M2 drug inhibitors interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins (Thomaston et al. 2018).

M2 colocalized and interacted with MAVS (mitochondrial antiviral signaling protein) on mitochondria, and positively regulated MAVS-mediated innate immunity. It induces reactive oxygen species (ROS) production that is required for activation of macroautophagy/autophagy and enhancement of MAVS signaling. The proton channel activity of M2 protein is essential for ROS production and antagonizing the autophagy pathway to control MAVS aggregation, thereby enhancing MAVS signal activity. Thus, M2 modulates host antiviral immunity (Wang et al. 2019).

Diazabicyclooctane derivatives with a constant charge of +2 block proton diffusion through the M2 ion channel (Vorobjev 2020). Two types of diazabicyclooctane derivatives were analyzed for binding to the M2 channel. An optimal structure was determined for a blocker to most efficiently block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport in addition to a steric barrier.

The generalized transport reaction catalyzed by the M2 channel is:

H+ (out) H+ (in).

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