M2 proton channel
||It has been suggested that Influenza virus matrix protein 2 be merged into this article. (Discuss) Proposed since March 2014.|
The M2 protein is a proton-selective ion channel protein, integral in the viral envelope of the influenza A virus. The channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds. It is activated by low pH. M2 protein is encoded on the seventh RNA segment together with the matrix protein M1. Proton conductance by the M2 protein in influenza A is essential for viral replication.
In influenza A virus M2 protein unit consists of three protein segments comprising 97 amino acid residues: (i) an extracellular N-terminal domain (residues 1–23); (ii) a trans membrane (TM) domain (residues 24–46); (iii) an intracellular C-terminal domain (residues 47–97). The TM domain forms the pore of the ion channel. The important residues are the imidazole of His37 (pH sensor) and the indole of Trp41 (gate). This domain is the target of the anti influenza drugs, Amantadine and its methyl derivative Rimantadine. The first 17 residues of the M2 cytoplasmic tail form a highly conserved amphipathic helix  The amphipathic helix residues (46–62) within the cytoplasmic tail play role in virus budding and assembly. The influenza virus utilizes these amphipathic helices in M2 to alter membrane curvature at the budding neck of the virus in a cholesterol dependent manner. The residues 70–77 of cytoplasmic tail are important for binding to M1 and for the efficient production of infectious virus particles. This region also contains a caveolin binding domain (CBD). The C-terminal end of the channel extends into a loop (residues 47–50) that connects the trans membrane domain to the C-terminal amphipathic helix. (46–62). Two different high-resolution structures of truncated forms of M2 have been reported: the crystal structure of a mutated form of the M2 transmembrane region (residues 22–46), as well as a longer version of the protein (residues 18–60) containing the transmembrane region and a segment of the C-terminal domain as studied by Nuclear magnetic resonance(NMR)  The two structures also suggest different binding sites for the adamantane class of anti-influenza drugs. According to the low pH crystal structure a single molecule of Amantadine binds in the middle of the pore, surrounded by residues Val27, Ala30, Ser31 and Gly34. In contrast, the NMR structure showed four Rimantadine molecules bind to the lipid facing outer surface of the pore, interacting with residues Asp44 and Arg45. However, a recent solid state NMR spectroscopy structure shows that the M2 channel has two binding sites for Amantadine, one high affinity site is in the N terminal lumen, and a second low affinity site on the C terminal protein surface.
M2 protein of Influenza B
The M2 protein of influenza B is 109 residue long, homo tetramer and is a functional homolog of influenza A protein. There is almost no sequence homology between influenza AM2 and BM2 except for the HXXXW sequence motif in the TM domain that is essential for channel function.Its proton conductance pH profile is similar to that of AM2. However, the BM2 channel activity is higher than that of AM2, and the BM2 activity is completely insensitive to Amantadine and Rimantadine.
The M2 protein is a low pH gated channel. It has a very low conductance rate of 10^4 protons per second compared to other classical proton channels. Lowering the pH from 8.2 to 4.2 increases the proton conductance by 50 folds. 
The M2 ion channel of both influenza A and B are highly selective for protons. Histidine residues at position 37 are responsible for this proton selectivity and pH modulation. When His37 is replaced with glycine, alanine, glutamic acid, serine or threonine, the proton selective activity is lost and the mutant can transport Na+ and K+ ions also. When imidazole buffer is added to cells expressing mutant proteins,the ion selectivity can be restored partially.
The M2 protein has an important role in both the early and late replication cycle of the influenza A virus. The M2 proton channel maintains pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation.As virus enters the host cell by receptor mediated endocytosis, endosomal acidification occurs. This low pH activates the M2 channel. M2 now brings protons into the virion core. Acidification of virus interior, leads to weakening of electrostatic interaction and leads to dissociation between M1 (matrix protein) and viral ribo nucleo protein (RNP) complexes. Subsequent membrane fusion releases the uncoated RNPs into the cytoplasm which is imported to the nucleus to start viral replication. After its synthesis within the infected host cell, M2 is inserted into the endoplasmic reticulum (ER) and transported to the cell surface via trans-Golgi network (TGN). Within the acidic TGN, M2 transports H+ ions out of the lumen, and maintains hemagglutinin(HA) metastable configuration.  At its TGN localization, M2 protein's ion channel activity has been shown to effectively activate the NLRP3 inflammasome pathway. Other important functions of M2 are its role in formation of filamentous strains of influenza, membrane scission and the release of the budding virion. M2 stabilizes the virus budding site, and mutations of M2 that prevent its binding to M1 can impair filament formation at the site of budding.
Inhibition and resistance
The function of the M2 channel can be inhibited by antiviral drugs amantadine and rimantadine, which then blocks the virus from taking over the host cell. The molecule of the drug binds to the transmembrane region, sterically blocking the channel. This stops the protons from entering the virion, which then does not disintegrate. Two different sites for drug interaction have been proposed. One is a lipid-facing pocket between 2 adjacent transmembrane helices (around Asp-44), at which the drug binds and inhibits proton conductance allosterically. The other is inside the pore (around Ser-31), at which the drug directly blocks proton passage. However, the M2 gene is susceptible to mutations. When one of five amino acids in the transmembrane region gets suitably substituted, the virus becomes resistant to the existing M2 inhibitors.S31N mutation is responsible for more than 90% resistance to the drugs Amantadine and Rimantadine. As the mutations are relatively frequent, presence of the selection factors (e.g. using Amantadine for treatment of sick poultry) can lead to emergence of a resistant strain. The US CDC has released information stating that most strains are now resistant to the two drugs available, and their use should be discontinued.
Sources and notes
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- M2 protein, Influenza A virus at the US National Library of Medicine Medical Subject Headings (MeSH)