Analysis of the pore structure of the influenza A virus M-2 ion channel bythe substituted-cysteine accessibility method

The M-2 ion channel of influenza A virus is a small integral membrane prote in whose active form is a homotetramer with each polypeptide chain containi ng 96-amino-acid residues. To identify residues of the transmembrane (TM) d omain 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) wa s replaced by cysteine, The accessibility of the cysteine mutants to modifi cation by the sulfhydryl-specific reagents methane thiosulfonate ethylammon ium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular ap plication of MTSEA evoked decreases in the conductances measured from two m utants, 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 extra cellular end of the channel than Ala-30, indicating the pore may be wider n ear the extracellular opening. To investigate the accessibility of the cyst eine mutants to reagents applied intracellularly, oocytes were microinjecte d directly with reagents during recordings. The conductance of the M-2-W41C mutant was decreased by intracellular injection of a concentrated MTSET so lution. 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 con striction in the pore exists between residues 34 and 41 which prevents pass age of the MTS reagent. These findings are consistent with the proposed rol e for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman , M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301-11306, 1997).