Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus.
The influenza virus M2 proton-selective ion channel activity facilitates virus uncoating, a process that occurs in the acidic environment of the endosome. The M2 channel causes acidification of the interior of the virus particle, which results in viral protein-protein dissociation. The M2 protein is a homotetramer that contains in its aqueous pore a histidine residue (His-37) that acts as a selectivity filter and a tryptophan residue (Trp-41) that acts as a channel gate. Substitution of His-37 modifies M2 ion channel properties drastically. However, the results of such experiments are difficult to interpret because substitution of His-37 could cause gross structural changes to the channel pore. We described here experiments in which partial or, in some cases, full rescue of specific M2 ion channel properties of His-37 substitution mutants was achieved by addition of imidazole to the bathing medium. Chemical rescue was demonstrated for three histidine substitution mutant ion channels (M2-H37G, M2-H37S, and M2-H37T) and for two double mutants in which the Trp-41 channel gate was also mutated (H37G/W41Y and H37G/W41A). Currents of the M2-H37G mutant ion channel were inhibited by Cu(II), which has been shown to coordinate with His-37 in the wild-type channel. Chemical rescue was very specific for imidazole. Buffer molecules that were neutral when protonated (4-morpholineethanesulfonic acid and 3-morpholino-2-hydroxypropanesulfonic acid) did not rescue ion channel activity of the M2-H37G mutant ion channel, but 1-methylimidazole did provide partial rescue of function. These results were consistent with a model for proton transport through the pore of the wild-type channel in which the imidazole side chain of His-37 acted as an intermediate proton acceptor/donor group.
Venkataraman
Padmavati
P
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Lamb
Robert A
RA
Pinto
Lawrence H
LH
D017738:Alkanesulfonic Acids / Q000494:pharmacology; D000547:Amantadine / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D011992:Endosomes; D005838:Genotype; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D007477:Ions; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008956:Models, Chemical; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D013451:Sulfonic Acids / Q000494:pharmacology; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis; D000086082:Viroporin Proteins
D017738:Alkanesulfonic Acids; D000547:Amantadine; D002021:Buffers; D003300:Copper; D006639:Histidine; D007093:Imidazoles; D007477:Ions; D009025:Morpholines; D011522:Protons; D012333:RNA, Messenger; D013451:Sulfonic Acids; D014364:Tryptophan; D014763:Viral Matrix Proteins; C501862:3-morpholino-2-hydroxypropanesulfonic acid; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C004550:2-(N-morpholino)ethanesulfonic acid; C029899:imidazole