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.
Peterson
Emily
E
Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602 USA. epetey63@gmail.com
Ryser
Ted
T
Funk
Spencer
S
Inouye
Daniel
D
Sharma
Mukesh
M
Qin
Huajun
H
Cross
Timothy A
TA
Busath
David D
DD
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