The M2 proton channels of influenza A and B viruses.

SummaryofStructure-FunctionRelationshipoftheA/M2IonChannel TheavailablebiochemicalandsolidstateNMRdataindicatethatthetrans-membrane domain of the A/M2 protein is comprised of a four-helix bundlewithatiltofabout25degrees.TheseexperimentsarealsoconsistentwiththefunctionalstudiesthatshowedthatthatresiduesVal 27 ,Ala 30 ,Gly 34 ,His 37 ,andTrp 41 line the aqueous pore. Furthermore, these results indicate that His 37 formsabarriertolargemoleculesandthatTrp 41 functionsasagatethatcloseswithhighpH out .Functionalstudiesindicatethattheportionofthecytoplasmicdomain nearest the membrane is important for normal ion channel function,andsolidstateNMRstudiesindicatethatthestructureofthisdomainbringsitinto close proximity to the inner membrane leaflet. The most remarkableaspect of this channel is the observation that much of its functionality is pro-vided for by the His 37 and Trp 41 residues found in one turn of the transmem-brane helical bundle. InhibitionoftheA/M2IonChannel ThistopicisimportantbecauseantiviraldrugsthatinhibittheM2ionchan-nel also inhibit replication of the influenza A virus, and understanding themechanismofinhibitionwouldbehelpfulindevelopingbetterinhibitors.TheantiviraldrugamantadineanditsderivativerimantadineinhibitthereplicationoftheinfluenzaAvirusbutnotinfluenzaBvirus(39).AmantadineinhibitstheA/M2ionchannel(12,17,40)butnottheBM2channel(16),whichisconsist-ent with amantadine inhibiting influenza A virus but not influenza B virusreplication.Severallinesofevidenceindicatethatinhibitionofviralreplicationby amantadine results from the inhibition of A/M2 proton channel activity.Thefirstevidencecomesfromamantadine-resistantescapemutantvirusesinwhich the site of the mutation was mapped and found to lie in the A/M2transmembrane domain (41). When these escape mutation proteins wereexpressed in oocytes (17, 42) or mammalian cells (12, 18) their currents werefoundtobeinsensitivetoamantadine.Thesecondlineofevidencecomesfromconsideration of the virus life cycle. Viral uncoating requires acidification ofthe virus prior to fusion with the endosomal membrane (43–45), and the M2protein is capable of providing the needed acidification when the virion iscontained in the endosome. The viral genome encodes three integral mem-brane proteins (46), and only A/M2 is capable of proton transport. Thus,inhibition of the essential proton transport function of the A/M2 ion channelresults in inhibition of replication of the virus.Five observations, taken together, suggest a mechanism for inhibition byamantadine. 1) Two of the mutations that result in resistance to amantadineoccur on residues that have been found by cysteine scanning mutagenesis toline the aqueous pore. Both of these mutations are to residues that are lesshydrophobic(A30TandG34E)thanthenativeresidue(41).2)Inhibitionofthechannel occurs more readily when pH of the bathing medium is high (40). 3)AmantadineonlyinhibitswhenitisappliedtothemediumbathingtheN-ter-minal ectodomain and not when applied to the C-terminal cytoplasmic tail;injection of as much as 1 m