Structural and Thermodynamic Properties of Selective Ion Binding in a K+ Channel

Abstract

Thermodynamic measurements of ion binding to the Streptomyces lividans K+ channel were carried out using isothermal titration calorimetry, whereas atomic structures of ion-bound and ion-free conformations of the channel were characterized by x-ray crystallography. Here we use these assays to show that the ion radius dependence of selectivity stems from the channel's recognition of ion size (i.e., volume) rather than charge density. Ion size recognition is a function of the channel's ability to adopt a very specific conductive structure with larger ions (K+, Rb+, Cs+, and Ba2+) bound and not with smaller ions (Na+, Mg2+, and Ca2+). The formation of the conductive structure involves selectivity filter atoms that are in direct contact with bound ions as well as protein atoms surrounding the selectivity filter up to a distance of 15 Å from the ions. We conclude that ion selectivity in a K+ channel is a property of size-matched ion binding sites created by the protein structure.

On the selective ion binding hypothesis for potassium channels

“…The KcsA selectivity filter possesses two Ba 2þ binding sites, in S4 and S2 (toward upper plane of S2) (42). We suggest that a filter occupied by Ba 2þ ions, to a significant extent in S2, could have considerable influence on the binding of K þ and Na þ ions in the external lock-in site above S2.…”

“…The accepted experimental measurement of preferential binding of K þ over Na þ ions in the K þ channel selectivity filter comes from an analysis of Ba 2þ channel blocking single channel recordings (11,12). Ba 2þ blocks the channel inside the selectivity filter (41,42). The external binding of K þ or Na þ , at a site termed the "lock-in" site, can prevent Ba 2þ from escaping to the outside.…”

Transmembrane allosteric coupling of the gates in a potassium channel

“…We show that extensive regions of KcsA quite distal to ion binding exhibit NMR spectral shifts in response to low potassium concentration. Moreover, the loci of these changes are well correlated with regions that are hypothesized from crystal structures to be perturbed as the channel opens at low pH (5,6,24) or correlated to regions where mutations affect inactivation kinetics (6,22,23,25,33). Evidence shown here for changes specifically in the hinge region and the pH sensor lead to the suggestion that not only is the ion expelled but that the global conformational change is likely to be the opening of the channel, akin to the changes during activation at low pH.…”

“…Because they contact selectivity filter sites on adjacent strands, residues M96 and F103 are presumed to be directly sensitive to the ion occupancy of the selectivity filter and have been shown through mutation to have an important role in the allostery of this system (6,25). It has been hypothesized that In addition to the selectivity filter, pH gate, and hinge region, residues exhibiting aggregate chemical shift perturbations greater than 1 ppm (defined as the sum of the absolute chemical shift difference of all resonances in each residue) were found in several other key regions of the protein including parts of the S1 helix (I38, V39, G43), the pore helix (W67, S69, V70, E71), and residues along the loop connecting the selectivity filter to the S2 helix (D80, L81, Y82).…”

“…5B) (6,22,23,25,33). The amino acids affected by K + binding are compared with those that have been implicated in inactivation of the channel in Table S1 and Fig.…”