Ion selectivity of the KcsA channel: a perspective from multi-ion free energy landscapes

Abstract

Potassium (K(+)) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K(+) through cell membranes. Comprising five specific cation binding sites (S(0)-S(4)) formed by the backbone carbonyl groups of conserved residues common to all K(+) channels, the narrow selectivity filter allows fast conduction of K(+) while being highly selective for K(+) over Na(+). To extend our knowledge of the microscopic mechanism underlying selectivity in K(+) channels, we characterize the free energy landscapes governing the entry and translocation of a Na(+) or a K(+) from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K(+) in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K(+) or Na(+) in binding site S(0) or S(1) are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na(+) than for K(+), implying that the kinetics of ion conduction is slower when a Na(+) enters the pore. Third, the region corresponding to binding site S(2) near the center of the narrow pore emerges as the most selective for K(+) over Na(+). In particular, while there is a stable minimum for K(+) in site S(2), Na(+) faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S(2) in the selectivity filter of K(+) channels.

Figure 1

Schematic illustration of the sequence of events needed for the entry of one Na⁺ ion from the extracellular side into the selectivity filter of the channel filled with K⁺ ions and water molecules (W).

Figure 2

Instantaneous MD simulation structure of the selectivity filter of KcsA occupied by one Na⁺ and two K⁺ ions in the configuration [S₀,S₂,S₄]. This structure belongs to the Na⁺/K⁺/K⁺ system, whereas the K⁺/K⁺/K⁺ system has three K⁺ ions in the selectivity filter region (not shown). For clarity, only backbone atoms of the residues forming the selectivity filter are displayed and two out of four monomers are omitted. The corresponding residue names are listed on the right. Above the selectivity filter is the extracellular side of the channel and below is a cavity filled with water molecules. The Na⁺ ion (ion I₁, purple sphere) occupies binding site S₀ at the extracellular entrance. The two K⁺ ions (I₂ and I₃, green spheres) are located in the binding sites S₂ and S₄. Each of the binding sites S₁ and S₃ is occupied by a single water molecule separating the ions in the selectivity filter. The image was produced using the program VMD.

Figure 3

Contour plots of 2D PMFs calculated from the 3D PMFs of the K⁺/K⁺/K⁺ system (left PMF) with solely K⁺ ions in the selectivity filter and of the Na⁺/K⁺/K⁺ system (right PMF), where one K⁺ ion is replaced by a Na⁺ ion. Two neighboring contour levels differ by 1 kcal/mol. Low energy regions are depicted in blue and high energy regions are depicted in red. The coordinate Z₁ is the position of ion I₁ (K⁺ or Na⁺) along the Z axis of the system, which is parallel to the pore formed by the selectivity filter. The 3D PMFs are projected on 2D PMFs with the coordinates Z₁ and Z₂₃ according to Eq. (1). The coordinate Z₂₃ is the mean of the respective positions Z₂ and Z₃ of the lower two K⁺ ions. The zero position along the Z axis of the system is defined by the center of mass of the backbone atoms of the residues Thr75, Val76, Gly77, and Tyr78 (see Fig. 2). A comparison of both PMFs shows that the energy well at the lower left corner of the K⁺/K⁺/K⁺ system PMF has no counterpart in the Na⁺/K⁺/K⁺ system PMF. Instantaneous snapshots taken from the MD simulations are used to illustrate the configuration of the ions in the selectivity filter for selected areas in the contour plots: (a) [S₀,S₂,S₄], (b) [S₁,S₃,cavity], and (c) [S₂,S₄,cavity]. The selectivity filter images were produced using the program VMD.

Figure 4

Contour plots of 2D PMFs calculated from those regions of the 3D PMFs which belong to the lower left areas in the contour plots of Fig. 3. Two neighboring contour levels differ by 1 kcal/mol. Low energy regions are depicted in blue and high energy regions are depicted in red. The 3D PMFs are projected on 2D PMFs with the coordinates Z₁ and Z₂ according to Eq. (2). These are the most relevant coordinates in this region, because only ion I₁, i.e. K⁺ (left PMF) or Na⁺ (right PMF), and ion I₂ are in the selectivity filter, whereas ion I₃ is already in the cavity. Images of instantaneous MD simulation snapshots show the location of the ions relative to the selectivity filter for selected areas of the contour plots: (b) [S₁,S₃,cavity] and (c) [S₂,S₄,cavity]. There are two energy wells in the K⁺/K⁺/K⁺ system PMF, but only one in the Na⁺/K⁺/K⁺ system PMF. The lower left energy well in the K⁺/K⁺/K⁺ system PMF belongs to a situation where ion I₁ is located at binding site S₂ (see snapshot (c)). The corresponding area of the Na⁺/K⁺/K⁺ system PMF shows a steep energy increase instead. The selectivity filter images were produced using the program VMD.

Figure 5

1D PMFs for the coordinate Z₁ (K⁺ or Na⁺ ion) calculated from the 3D PMFs according to Eq. (3). The Na⁺ Z₁ coordinate is shifted by 0.7 Å relative to the K⁺ Z₁ coordinate. This overlays the main minima of the PMFs and makes it easier to compare them. The K⁺/K⁺/K⁺ system PMF has energy minima at the positions of the binding sites S₀, S₁, and S₂. The Na⁺/K⁺/K⁺ system PMF has energy minima at the positions of the binding sites S₀ and S₁, whereas no comparable minima can be found where binding site S₂ would be expected.

Figure 6

Average coordination numbers for ion I₁ at different ion positions Z₁ for special paths in the 3D PMF landscapes. The average numbers of water, carbonyl, and total oxygen atoms which are not further away than 3.2 Å from ion I₁ in the K⁺/K⁺/K⁺ system and 2.8 Å in the Na⁺/K⁺/K⁺ system are shown.