Biophys J, March 2000, p. 1335-1348, Vol. 78, No. 3

Protonation of Lysine Residues Inverts Cation/Anion Selectivity in a Model Channel

Vitali Borisenko,^* Mark S. P. Sansom, and G. Andrew Woolley^*

 ^*Department of Chemistry, University of Toronto, Toronto M5S 3H6, Canada, and  Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

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.

Biophys J, March 2000, p. 1335-1348, Vol. 78, No. 3
© 2000 by the Biophysical Society   0006-3495/00/03/1335/14  $2.00

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