Measurement of Selectivity Filter Dynamics in Selective and Non-Selective NaK Channel Variants

Abstract

K+ channel selectivity is governed by the selectivity filter (SF), a region of the pore lined by backbone carbonyl moieties that contacts permeating ions. There is ongoing debate as to the relative importance of various SF features in producing K+-selectivity, such as ligand type, number of ion binding sites, structural organization of the binding sites, and rigidity of the SF structure. NaK, a non-selective ion channel from Bacillus cereus bears two of the four canonical K+ binding sites and can be made K+-selective by mutating the SF sequence to the canonical K+ SF sequence. This mutant, NaK2K, has an SF structure virtually identical to that of bona fide K+ channels, inspiring the hypothesis that four contiguous ion binding sites are necessary to confer K+ selectivity. However, when Y55 is mutated to F in NaK2K (NaK2K-Y55F), the SF structure remains the same as seen by X-ray crystallography, but the channel is not selective for K+ over Na+. The authors speculate that the NaK2K-Y55F SF is more dynamic in solution than the NaK2K SF, resulting in reduced binding site number and loss of K+ selectivity. This hypothesis can be tested by performing NMR spin relaxation measurements that are sensitive to ps-ns timescale dynamics. Data collected for NaK suggests that dynamics vary across the SF; the extracellular mouth is more dynamic than the intracellular base where the two conserved binding sites are located. It is expected that the dynamics of NaK2K-Y55F will mirror those of NaK, rather than NaK2K.