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, WI. 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 PK+/PCl- = 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 PK+/PCl- = 4, In contrast, native alamethicin-Q18 channels (with Gin in place of Lys at position 18) were cation-selective (PK+/PCl- = 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.