Functional reconstitution of influenza A M2(22-62).
Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K(+)] gradient-induced membrane potential and [H(+)] gradient), M2(22-62) showed a transport rate of 78 H(+)/tetramer-s (pH(o) 6.0, pH(i) 8.0, nominal V(m)=-114 mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05-1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K(+) flux of 0.1K(+)/tetramer-s, and also demonstrated that the K(+) permeability, relative to H(+), was 2.8 × 10(-6). Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H(+) selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pK(a) of 6.
Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602 USA. epetey63@gmail.com
Emily
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Peterson
Ted
T
Ryser
Spencer
S
Funk
Daniel
D
Inouye
Mukesh
M
Sharma
Huajun
H
Qin
Timothy A
TA
Cross
David D
DD
Busath
D000547:Amantadine / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014634:Valinomycin / Q000494:pharmacology; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000086082:Viroporin Proteins
D000547:Amantadine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D007473:Ion Channels; D008081:Liposomes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014634:Valinomycin; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins