Differential binding of monovalent cations to KcsA: Deciphering the mechanisms of potassium channel selectivity

Abstract

This work explores whether the ion selectivity and permeation properties of a model potassium channel, KcsA, could be explained based on ion binding features. Non-permeant Na or Li bind with low affinity (millimolar K's) to a single set of sites contributed by the S1 and S4 sites seen at the selectivity filter in the KcsA crystal structure. Conversely, permeant K, Rb, Tl and even Cs bind to two different sets of sites as their concentration increases, consistent with crystallographic evidence on the ability of permeant species to induce concentration-dependent transitions between conformational states (non-conductive and conductive) of the channel's selectivity filter. The first set of such sites, assigned also to the crystallographic S1 and S4 sites, shows similarly high affinities for all permeant species (micromolar K's), thus, securing displacement of potentially competing non-permeant cations. The second set of sites, available only to permeant cations upon the transition to the conductive filter conformation, shows low affinity (millimolar K's), thus, favoring cation dissociation and permeation and results from the contribution of all S1 through S4 crystallographic sites. The differences in affinities between permeant and non-permeant cations and the similarities in binding behavior within each of these two groups, correlate fully with their permeabilities relative to K, suggesting that binding is an important determinant of the channel's ion selectivity. Conversely, the complexity observed in permeation features cannot be explained just in terms of binding and likely relates to reported differences in the occupancy of the S2 and S3 sites by the permeant cations.

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel

“…The second set of sites i) results from the contribution of all S1 through S4 crystallographic sites, ii) is available only to permeant cations when the filter is in the conductive conformation and iii) shows low affinity (millimolar K D 's), thus, favoring cation dissociation and permeation. The differences in affinities between permeant and non-permeant cations and the similarities in binding behavior within each group correlate fully with their permeabilities relative to K + and therefore, it was concluded that cation binding may be an important determinant of ion selectivity (31). Here we report new evidences that greatly reinforce such hypothesis.…”

“…Figure 2 illustrates the first of such approaches in which the channel in a nonconductive state caused by 100 mM Na + , was titrated with the permeant K + , Rb + or Cs + . Similarly to that observed under non-competitive conditions (35), i.e., where titrations started with a channel prepared in just 1.5 mM Na + , the addition of increasing concentrations of any of the permeant cations, resulted in a large thermal stabilization of the channel protein ( Figure 2A, B and C). This indicates that all such permeant cations can efficiently displace Na + from binding to the channel pore; although K + and Rb + were always more efficient in this regard than Cs + .…”

“…This indicates that all such permeant cations can efficiently displace Na + from binding to the channel pore; although K + and Rb + were always more efficient in this regard than Cs + . Nonetheless, instead of the characteristic two binding events reported for permeant cations under non-competitive conditions (35), three different binding events are now clearly detected in the titration curves ( Figure 2D, E and F). This was not unexpected because, as described in the previous paragraph, the S1 and S4 sites have different affinities for Na + , which must be competitively displaced from both sites by the incoming permeant cation.…”

“…In order to test such possibility, we determined those critical concentrations in dilute solution conditions for the three permeant cations used here. K D values of 2.5, 3.4 and 15 mM, for K + , Rb + and Cs + , respectively, were determined for the low affinity binding event inducing the conformational change in the selectivity filter (35), which are similar to those determined from crystallography (7). Therefore, in the experiments described below, we use K + at concentrations within such critical range to give raise to conductive or nonconductive filter states in which to test competitive Na + binding.…”

“…These latter findings support the notion from X-ray studies that the selectivity filter itself and/or its immediate surroundings are the site(s) involved both in cation binding and in the cation-induced protein conformational changes. Third, it was found that fluorescence monitoring of thermal denaturation of KcsA, which includes the dissociation of the tetrameric protein into its subunits and their partial unfolding (26,(28)(29)(30), provides an experimental observable, the apparent t m of the native to denatured thermal transition, ideally suited to study cation binding (26,31). The nonpermeant species Na + or Li + bind with low affinity (millimolar K D 's) to a single set of binding sites contributed by the crystallographic S1 and S4 sites.…”