The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer-1.2 aA (1.2 ؋ 10 ؊18 A) at neutral pH and 2.7 to 4.1 aA at pH 5.7-which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na ؉ or K ؉ probes. Even low-pH-activated M2 did not conduct Na ؉ and K ؉ . The proton selectivity of M2 was estimated to be at least 3 ؋ 10 6 (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.Viruses such as influenza virus (A, B, and C) and human immunodeficiency virus have evolved ion channel proteins that assist their invasion of the host cell or their egress from the biosynthetic machinery (for reviews, see references 14, 19, and 20). As the first viral ion channel protein to be discovered, as well as the target of the classic antivirals amantadine and rimantadine, the influenza A virus M2 protein has become the paradigm of this new class of viral proteins. For uncoating, the virus is dependent on the acidity of the endosome, but to protect the maturation of acid-sensitive hemagglutinin (HA [of the H7 and H5 subtypes]), it needs to avoid the low pH in the trans-Golgi network (TGN). The M2 protein fulfills both of these functions by equilibrating membrane pH gradients (14).We developed procedures for the expression, isolation, and reconstitution of the M2 protein into liposomes, as well as a functional assay, demonstrating that M2 translocates protons in a rimantadine-sensitive manner (36). We now present quantitative data on single-channel conductance and ion selectivity determined in this system.The initial report (28) on the electrophysiology of the M2 protein and several later studies represented M2 as an acidactivated sodium ion or unspecific monovalent cation channel (37,41,42). On the other hand, whole-cell recordings of M2-expressing MEL cells confirmed our observation of proton conductiv...

The influenza-virus M2 protein has proton channel activity required for virus uncoating and maturation of hemagglutinin (HA) through low-pH compartments. The proton channel is cytotoxic in heterologous expression systems and can be blocked with rimantadine. In an independent, rimantadine-resistant function, M2, interacting with the M1 protein, controls the shape of virus particles. These bud from cholesterol-rich membrane rafts where viral glycoproteins and matrix (M1)/RNP complexes assemble. We demonstrate that M2 preparations from influenza virus-infected cells and from a baculovirus expression system contain 0.5-0.9 molecules of cholesterol per monomer. Sequence analyses of the membrane-proximal M2 endodomain reveal interfacial hydrophobicity, a cholesterol-binding motif first identified in peripheral benzodiazepine receptor and human immunodeficiency virus gp41, and an overlapping phosphatidylinositol 4,5-bisphosphate-binding motif. M2 induced rimantadine-reversible cytotoxicity in intrinsically cholesterol-free E. coli, and purified E. coli-expressed M2 functionally reconstituted into cholesterol-free liposomes supported rimantadine-sensitive proton translocation. Therefore, cholesterol was nonessential for M2 ion-channel function and cytotoxicity and for the effect of rimantadine. Only about 5-8% of both M2 preparations, regardless of cholesterol content, associated with detergent-resistant membranes. Cholesterol affinity and palmitoylation, in combination with a short transmembrane segment suggest M2 is a peripheral raft protein. Preference for the raft/non-raft interface may determine colocalization with HA during apical transport, the low level of M2 incorporated into the viral envelope and its undisclosed role in virus budding for which a model is presented. M2 may promote clustering and merger of rafts and the pinching-off (fission) of virus particles.

The influenza virus M2 protein, target of the antiviral drugs amantadine and rimantadine, forms a proton channel which functions during virus uncoating and maturation by modifying the pH in virions as well as in trans-Golgi vesicles. We studied the influence of different ionic gradients on the inhibition of the proton translocation activity of isolated, baculovirus-expressed M2 protein reconstituted into liposomes. Two distinct patterns of inhibition were observed. A group of amphiphilic amines including amantadine, cyclooctylamine and rimantadine inhibited M2 effectively in the presence of physiological Na M concentrations. The 10-fold greater activity of rimantadine over amantadine and the 100-fold stronger effect of cyclooctylamine compared to cyclopentylamine matched the relative activities in influenza virus-infected cells. A com-