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Structural and thermodynamic properties of selective ion binding in a K+ channel - PubMed

Comparative Study

Structural and thermodynamic properties of selective ion binding in a K+ channel

Steve W Lockless et al. PLoS Biol. 2007 May.

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 Ba(2+)) bound and not with smaller ions (Na(+), Mg(2+), and Ca(2+)). 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 A 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.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. K+ Ion Binding Sites within the KcsA K+ Channel

(A) A ribbon representation of KcsA with two of the four subunits is shown; the subunits closest to and furthest from the viewer are removed for clarity. K+ ions (in green) are located within the selectivity filter (orange) and in the water-filled cavity. The gray lines indicate the presumed interior and exterior membrane boundaries. (B) A stick representation of the selectivity filter containing four K+ ion-binding sites (S1–S4). Each K+ ion-binding site is composed of eight oxygen atoms made from the K+ channel TVGYG signature sequence. The figure was made using PyMOL [33].

Figure 2
Figure 2. ITC Titration of Alkali Metal Cations Binding to KcsA

(A) A KCl solution is titrated into a solution containing KcsA. Each downward deflection corresponds to one injection. The area represents the heat exchanged. (B) The total heat exchanged during each injection is fit to a single-site binding isotherm with K D and ΔH° as independent parameters, where K D = 0.41 mM and ΔH° = −1.4 kcal/mol. Similar values for K D and ΔH° are obtained using different protein concentrations varied over a 5-fold range. The inset shows the same data represented as the fraction of ion-bound KcsA as a function of [KCl]. (C) A KCl solution titrated into KcsA in a background solution of LiCl instead of NaCl (as is used in Figure 2A) shows heat from ion binding. (D) A NaCl solution titrated into KcsA in the background of LiCl shows only the heat of diluting NaCl without binding to KcsA.

Figure 3
Figure 3. A Conformational Change Underlies the Enthalpy of Ion Binding

(A) The KcsA selectivity filter exists in two conformations that depend on whether one or two ions are bound. The collapsed conformation (left) has one ion distributed over two sites, whereas the conductive conformation (right) has two ions distributed over four sites. Amino acids E71 to D80 are shown in a stick diagram (from PDB 1K4D and PDB 1K4C) encompassing the selectivity filter (T75-G79) and surrounding residues. (B) This stereo-view compares the KcsA-M96V structure (gray) to the KcsA-wt collapsed structure (red) and shows that the M96V mutant remains in the collapsed conformation even at 300 mM KCl. (C) An ITC titration of wild-type KcsA with 400 mM KCl in the syringe shows most of the ion binding occurs within the first few injections. (D) An ITC titration of the M96V mutant with KCl shows no K+ binding.

Figure 4
Figure 4. Propagated Residue Displacements Correlate with Ion Binding and Co-Evolving Positions

(A) A comparison of the collapsed and conductive conformations of KcsA is shown. The collapsed Na+-bound structure is shown in red, whereas the conductive K+-, Rb+-, or Cs+- bound structures are shown in various shades of blue. This image is a slice through the S2 ion-binding site as viewed from the extracellular side of the membrane looking down the pore. (B) A stereo-view of the highly conserved selectivity filter (orange) and conserved plus co-evolving (yellow) positions of K+ channels is shown as a stick representation mapped onto the ribbon representation (blue) of KcsA. The brown surface envelops the conserved and co-evolving positions. This view is from the extracellular side of the membrane looking down the pore.

Figure 5
Figure 5. ITC Titration of Alkaline Earth Metal Cations Binding to KcsA

(A) A BaCl2 solution is titrated into a solution containing KcsA. (B) The total heat exchanged is fit to a single binding isotherm with K D and ΔH° as independent variables. (C) A CaCl2 solution titrated into a solution containing KcsA shows no ion binding.

Figure 6
Figure 6. Structure of the Selectivity Filter with Ba2+ Bound Inside

(A) A 2Fo-Fc electron density map (colored in blue and contoured at 2 sigma) of the selectivity filter region show two diagonally opposing subunits. Ba2+ ions (in magenta) and H2O (in red) are shown as spheres within the filter. The anomalous difference density map is shown in magenta as a fine mesh (contoured at 10 sigma) and was used to identify the Ba2+ ions. (B) A stick representation shows a comparison between the selectivity filter of the Ba2+ bound structure (yellow) with the collapsed (red) and conductive (blue) K+ bound structures. (C) Comparing the Ba2+- bound (yellow) and conductive conformation (blue) structures shows that the Ba2+- bound structure adopts the conductive conformation at distant sites along the plane of aromatic amino acids surrounding the selectivity filter.

Figure 7
Figure 7. Ion-Binding Model

The four-state ion-binding scheme is shown, from left to right: non-conductive conformation without an ion (N), non-conductive conformation with a single ion (NX), conductive conformation with a single ion (CX), and conductive conformation with two ions (CX2) with equilibrium constants K 0, K 1, and K 2.

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