The influenza virus M 2 proton-selective ion channel is known to be essential for acidifying the interior of virions during virus uncoating in the lumen of endosomes. The M 2 protein is a homotetramer that contains four 19-residue transmembrane (TM) domains. These TM domains are multifunctional, because they contain the channel pore and also anchor the protein in membranes. The M 2 protein is gated by pH, and thus we have measured pH-gated currents, the accessibility of the pore to Cu 2Ű , and the effect of a protein-modifying reagent for a series of TM domain mutant M 2 proteins. The results indicate that gating of the M 2 ion channel is governed by a single side chain at residue 41 of the TM domain and that this property is mediated by an indole moiety. Unlike many ion channels where the gate is formed by a whole segment of a protein, our data suggest a model of striking simplicity for the M 2 ion channel protein, with the side chain of Trp 41 blocking the pore of the M 2 channel when pH out is high and with this side chain leaving the pore when pH out is low. Thus, the Trp 41 side chain acts as the gate that opens and closes the pore.The prediction that the influenza A virus M 2 protein has a proton-selective ion channel activity (Refs. 1 and 2 and reviewed in Ref.3) arose from a coupling of various observations on the life cycle of influenza virus. The M 2 protein is an integral membrane protein that is expressed at the plasma membrane of influenza virus-infected cells and is incorporated in small amounts into budding virions (4, 5). Studies on the mechanism of action of the anti-viral drug, amantadine (1-aminoadamantine hydrochloride), indicated that viral escape mutants resistant to the drug mapped to the transmembrane (TM) 1 domain of the M 2 protein (6) and that amantadine affected two steps in the life cycle, virus uncoating and virus maturation. The effect of amantadine on inhibition of uncoating is general to all strains of influenza A virus (7, 8) (reviewed in Refs. 3, 9 -11). 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 (M1) and the ribonucleoprotein (RNP) core (7,(12)(13)(14)(15)). In the presence of amantadine, influenza virus uncoating is incomplete, because the M1 protein is not released from the RNPs and the RNPs fail to enter the nucleus. Normally, influenza virus RNPs are transcribed and replicated in the nucleus (reviewed in Ref. 10). For some influenza virus subtypes, amantadine inhibits a "late" step in virus replication. The M 2 ion channel activity is activated during transport of the M 2 protein through the exocytic pathway; this ion channel activity raises the lumenal pH of the trans Golgi network (TGN), equilibrating pH with that of the cytoplasm (1, 17-22). Thus, the intralumenal pH of the TGN i...