Functional reconstitution of influenza A M2(22–62)

• Published in: Biochimica et biophysica acta 2011-02, Vol.1808 (2), p.516-521
• Main Authors: Peterson, Emily, Ryser, Ted, Funk, Spencer, Inouye, Daniel, Sharma, Mukesh, Qin, Huajun, Cross, Timothy A., Busath, David D.
• Format: Article
• Language: English
• Subjects: Acid activation; Amantadine - pharmacology; Carbonyl Cyanide m-Chlorophenyl Hydrazone - pharmacology; Hydrogen-Ion Concentration; Influenza A virus; Influenza A virus - chemistry; Influenza A virus - genetics; Influenza A virus - metabolism; Ion Channels - chemistry; Ion Channels - genetics; Ion Channels - metabolism; Kinetics; Liposome assay; Liposomes; M2 protein; Membrane Potentials; Membrane protein; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Specific transport activity; Valinomycin - pharmacology; Viral Matrix Proteins - chemistry; Viral Matrix Proteins - genetics; Viral Matrix Proteins - metabolism
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2010.10.010

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 0.1 K +/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.