Amino Acid Substitutions for T75 in KCSA Alter Ion Selectivity

Abstract

The K+/Na+ ion selectivity of the bacterial ion channel KcsA is ≈400. We have shown from non-equilibrium molecular dynamics (MD) simulations that entry of the ions into the selectivity filter of KcsA is associated with a free energy barrier that is approximately 3.7 kcal/mol higher for Na+ than for K+, and that this free energy barrier effectively excludes Na+ from the selectivity filter of KcsA. Na+ is stabilized just outside the selectivity filter of KcsA in the water-filled central cavity (vestibule) by interactions with the side chain of the T75 residues of each KcsA subunit. In silico amino acid substitutions were made for T75 in KcsA to serine, valine, and cysteine in order to examine the consequences of replacing the side chain groups in threonine on Na+ and K+ permeation. Threonine has side chain methyl and hydroxyl groups, and serine has a proton and a hydroxyl group, valine has two methyl groups, and cysteine has a proton and a thiol group in their side chains. Single Na+ or K+ ions were pulled through the wild type and mutant channels using a step-wise pulling protocol and Jarzynski's Equality to obtain work distributions and free energy values for each ion moving through the channel. The simulations showed that valine and serine excluded both Na+ and K+ from the selectivity filter under conditions where the wild type channel excluded only Na+; however, cysteine allowed both ions to enter the selectivity filter. These results suggest that mutant KcsA channels having serine, valine, or cysteine in the position of T75 will have reduced K+/Na+ selectivity. The simulations also indicate that differential dehydration of the ions is not correlated with changes in ion selectivity in the mutants.