Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel.
The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21-61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22-46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pK(a) values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.
Ma
Chunlong
C
Departments of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208-3500, USA.
Polishchuk
Alexei L
AL
Ohigashi
Yuki
Y
Stouffer
Amanda L
AL
Schön
Arne
A
Magavern
Emma
E
Jing
Xianghong
X
Lear
James D
JD
Freire
Ernesto
E
Lamb
Robert A
RA
DeGrado
William F
WF
Pinto
Lawrence H
LH
D000547:Amantadine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015259:Dopamine Agents / Q000494:pharmacology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D055503:Protein Multimerization; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014981:Xenopus; D000086082:Viroporin Proteins
D000547:Amantadine; D015259:Dopamine Agents; D007473:Ion Channels; D010446:Peptide Fragments; D011522:Protons; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins