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.
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