Structure–function relationship of the M 2 ion channel of influenza A virus

Abstract

The use of oligomers comprised of amantadine-sensitive and -resistant forms demonstrated that the active oligomeric state of the channel is a tetramer. Cysteine scanning mutagenesis followed by evaluation of the ability of sulfhydryl-specific reagents to inhibit the channel demonstrated that each monomer is a coiled coil and that the pore-lining residues are Val-27, Ala-30, Gly-34, His-37 and Trp-41. Under oxidizing conditions, mutant proteins containing cysteine at or close to these residues form dimers, but do so less readily at low pH for residues near #41, supporting the notion that a pH-induced conformational change occurs in this region of the protein. A functional change in state was detected by comparing the efflux of H + from acid-loaded cells expressing M 2 protein with those treated with the protonophore FCCP: the efflux was high for FCCP-treated cells, but not M 2-expressing cells when pH out was elevated. The current of the wild-type protein is carried by H + and is increased by low pH out, but replacement of the His-37 results in pH-independent currents of other ions as well, suggesting that the selectivity and activation of the channel might result from the action of His-37. The C-terminus is needed for sustained function of the channel, as truncation mutants are abnormal and that they pass H + for only a short time.