Protonation of lysine residues inverts cation/anion selectivity in a model channel.
A dimeric alamethicin analog with lysine at position 18 in the sequence (alm-K18) was previously shown to form stable anion-selective channels in membranes at pH 7.0 [Starostin, A. V., R. Butan, V. Borisenko, D. A. James, H. Wenschuh, M. S. Sansom, and G. A. Woolley. 1999. Biochemistry. 38:6144-6150]. To probe the charge state of the conducting channel and how this might influence cation versus anion selectivity, we performed a series of single-channel selectivity measurements at different pH values. At pH 7.0 and below, only anion-selective channels were found with P(K(+))/P(Cl(-)) = 0. 25. From pH 8-10, a mixture of anion-selective, non-selective, and cation-selective channels was found. At pH > 11 only cation-selective channels were found with P(K(+))/P(Cl(-)) = 4. In contrast, native alamethicin-Q18 channels (with Gln in place of Lys at position 18) were cation-selective (P(K(+))/P(Cl(-)) = 4) at all pH values. Continuum electrostatics calculations were then carried out using an octameric model of the alm-K18 channel embedded in a low dielectric slab to simulate a membrane. Although the calculations can account for the apparent pK(a) of the channel, they fail to correctly predict the degree of selectivity. Although a switch from cation- to anion-selectivity as the channel becomes protonated is indicated, the degree of anion-selectivity is severely overestimated, suggesting that the continuum approach does not adequately represent some aspect of the electrostatics of permeation in these channels. Side-chain conformational changes upon protonation, conformational changes, and deprotonation caused by permeating cations and counterion binding by lysine residues upon protonation are considered as possible sources of the overestimation.
Borisenko
V
V
Department of Chemistry, University of Toronto, Toronto M5S 3H6, Canada.
Sansom
M S
MS
Woolley
G A
GA
D000408:Alamethicin / Q000031:analogs & derivatives* / Q000737:chemistry*; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008239:Lysine; D008564:Membrane Potentials; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D011137:Polystyrenes; D055672:Static Electricity; D013499:Surface Properties
D007473:Ion Channels; D011137:Polystyrenes; D000408:Alamethicin; D008239:Lysine