The discovery that the influenza A virus M 2 protein has proton-selective ion channel activity stemmed from an understanding of the life cycle of influenza virus and the two steps in the life cycle that are inhibited by the antiviral drug amantadine (reviewed in reference 14). Direct evidence that the M 2 protein has a low-pH-activated, proton-selective conductance was obtained by expressing the M 2 protein in oocytes of Xenopus laevis (20,21,47,53,64,65,67) or mammalian cells (6,40,41,66). M 2 -specific cell surface currents were measured, and they were found to be specifically blocked by amantadine. Furthermore, when either peptides corresponding to the M 2 transmembrane (TM) domain or purified M 2 protein was incorporated into planar bilayers, an amantadine-sensitive current was measured (11,36,52,63).Amantadine inhibits the early step of uncoating of influenza virus in endosomes (reviewed in references 14, 31, 32, and 34). When a virion has entered the cell by receptor-mediated endocytosis and the virus particle is in the acidic environment of the endosomal lumen, the M 2 ion channel is activated and conducts protons across the viral membrane. The lowered internal virion pH is thought to weaken protein-protein interactions between the viral matrix protein (M 1 ) and the ribonucleoprotein (RNP) core (4,5,38,72,73; reviewed in reference 18

The homotetrameric M 2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M 2 transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine. A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals. Thus, membrane currents of oocytes of Xenopus laevis expressing the M 2 protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu 2؉ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (K diss ϳ2 M) that lies deep within the pore and shows rather slow-binding kinetics (k on ‫؍‬ 18.6 ؎ 0.9 M ؊1 s ؊1 ). The pH dependence of the interaction with the high affinity Cu 2؉ -binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu 2؉ could be prevented by prior application of the reversible blocker of M 2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M 2 . Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu 2؉ . Binding of Cu 2؉ to the high affinity site resulted in an approximately equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu 2؉ and was only partially inhibited by 1 mM Ni 2؉ , Pt 2؉ , and Zn 2؉. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel.The M 2 protein of influenza A virus is thought to function as an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes. In addition, in influenza virus-infected cells the M 2 protein causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (reviewed in Refs. 1 and 2). The M 2 protein contains a 24-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues which acts as a transmembrane domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (3). Chemical cross-linking studies showed the M 2 protein to be minimally a homotetramer (4 -6), and statistical analysis of the ion channel activity of mixed...

The M 2 ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M 2 -A30C and M 2 -G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M 2 -W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M 2 -G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken The M 2 ion channel protein of influenza A virus has a high selectivity for protons (3,8,17,21,24). Even though the M 2 protein is only a minor component of the viral envelope (30), the ion channel activity is nonetheless essential in the life cycle of the virus. Influenza virus enters cells via endocytosis, and in the low-pH environment found in the endosomal compartment, the M 2 ion channel is activated (reviewed in reference 14). Protons flow into the virion, acidifying the virion interior, a pH change which is thought to weaken protein-protein interactions between the viral matrix protein and the ribonucleoprotein (RNP) complex (reviewed in references 10 and 14), a prerequisite in the uncoating process. A considerable body of evidence indicates the M 2 ion channel is inhibited by the antiviral drug amantadine (3, 21, 29). In virus-infected cells treated with amantadine, the viral matrix protein fails to dissociate from the RNP, and import of the RNA into the nucleus does not occur. Hence, the RNPs cannot undergo replication in the nucleus and the viral life cycle is blocked (reviewed in references 10, 14, and 15).The mature M 2 protein is 96 amino acid residues (the Nterminal methionine is cleaved [27]), and it spans the membrane once: it has 23 extracellular residues, a transmembrane (TM) domain of 19 residues, and a 54-residue cytoplasmic...

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