Conformational changes in the selectivity filter of the open-state KcsA channel: an energy minimization study

Abstract

Potassium channels switch between closed and open conformations and selectively conduct K(+) ions. There are at least two gates. The TM2 bundle at the intracellular site is the primary gate of KcsA, and rearrangements at the selectivity filter (SF) act as the second gate. The SF blocks ion flow via an inactivation process similar to C-type inactivation of voltage-gated K(+) channels. We recently generated the open-state conformation of the KcsA channel. We found no major, possibly inactivating, structural changes in the SF associated with this massive inner-pore rearrangement, which suggests that the gates might act independently. Here we energy-minimize the open state of wild-type and mutant KcsA, validating in silico structures of energy-minimized SFs by comparison with crystallographic structures, and use these data to gain insight into how mutation, ion depletion, and K(+) to Na(+) substitution influence SF conformation. Both E71 or D80 protonations/mutations and the presence/absence of protein-buried water molecule(s) modify the H-bonding network stabilizing the P-loops, spawning numerous SF conformations. We find that the inactivated state corresponds to conformations with a partially unoccupied or an entirely empty SF. These structures, involving modifications in all four P-loops, are stabilized by H-bonds between amide H and carbonyl O atoms from adjacent P-loops, which block ion passage. The inner portions of the P-loops are more rigid than the outer parts. Changes are localized to the outer binding sites, with innermost site S4 persisting in the inactivated state. Strong binding by Na(+) locally contracts the SF around Na(+), releasing ligands that do not participate in Na(+) coordination, and occluding the permeation pathway. K(+) selectivity primarily appears to arise from the inability of the SF to completely dehydrate Na(+) ions due to basic structural differences between liquid water and the "quasi-liquid" SF matrix.

FIGURE 1

Side view of the central simulation cell showing open-state KcsA, 3 K⁺ ions, 123 DPPC lipids, and ∼7000 waters (∼400 in the intracellular vestibule). Three KcsA subunits are shown in backbone representation in green, blue, and yellow colors. Lipid head groups are shown in native colors. For clarity, fronting lipids and one KcsA subunit are not displayed. K⁺ ions are depicted as pink spheres. Figs. 1–4 and 6 were generated using our MCICP code (21). (See online version for color Figs. 1–5.)

FIGURE 2

Conformations of the SF of closed- (high-[K⁺] structure, 1K4C in blue) and open-state (energy-minimized KcsA, native colors) structures for protonated E71 and deprotonated D80, (a) with and (b) without a water molecule behind the P-loop. The three K⁺ ions in the pore at their crystal locations are illustrated in blue. SF residues of only one subunit are displayed. Filter residues are labeled near their carbonyl ends. Backbone atoms are only shown for residues 72–79. Side chains of E71, D80, R89, and W67 (energy-minimized KcsA) are shown in green, with atoms participating in H-bonding shown in native colors.

FIGURE 3

Conformations of the SF of closed- (high-[K⁺] structure, 1K4C in blue) and open-state (energy-minimized KcsA, native colors) structures for both E71 and D80 ionized, (a and b) with and (c and d) without a water molecule behind the P-loop, respectively. The location of K⁺ ions and filter waters, the residue labeling, and the color scheme are as in Fig. 2.

FIGURE 4

Conformations of the SF of (a) closed low [K⁺] (1K4D, blue) and open-state energy-minimized E71p KcsA (native colors) and (b) closed nonflipped E71A KcsA (1ZWI, blue) and open-state energy-minimized E71A KcsA (native colors) structures. (a) Site S2 is vacant and two water molecules are located behind the P-loop; K⁺ (purple) occupies sites S1 and S4, with Na⁺ (green) in the cavity. (b) Blue spheres represent the cavity, and S1 and S3 binding sites of 1ZW1; in the minimized structure, K⁺ (purple) occupies these sites.

FIGURE 5

The energy-minimized structure of the four P-loops, void of K⁺ and waters, in the SF of (a) E71p KcsA and (b) E71A KcsA. Only the backbone (CA, C, O, N, and HN) atoms of the P-loops are shown (native colors). To provide the impression of depth, a fog effect is employed, with above plane atoms brightest and lower ones faded to a background color. H-bonds stabilizing these conformations are shown as dashed lines and distances are labeled. K⁺ occupies the cytoplasmic vestibule and waters are shown in the cytoplasmic vestibule and exterior mouth. The H-bonds between adjacent P-loops are shown in the enlarged insets with the view along the channel axis as seen (a) from the periplasm for E71p KcsA and (b) from the cytoplasm for E71A KcsA. The figure is drawn using VMD (28).

FIGURE 6

The energy-minimized SF loaded with Na⁺ for two protonation variants: (a) E71p and (b) both E71 and D80 charged. Waters, Na⁺, and carbonyl Os from the four P-loops are shown. To provide the impression of depth, the fog effect is employed, with above plane atoms brightest and lower ones faded to a background color. Sodium-oxygen bonds are shown as dashed lines and distances are labeled. One of the carbonyl Os of V76 in a has flipped. The distance between sodium and O atoms with labels 1, 2, and 3 in b is larger than 2.8 Å, and coordination is five-fold. The insets present enlarged pictures of fivefold coordination by (a) water O and a ring of four carbonyl Os and (b) five carbonyl Os.