Common mechanism of pore opening shared by five different potassium channels

Abstract

A fundamental question associated with the function of ion channels is the conformational changes that allow for reversibly opening/occluding the pore through which the cations permeate. The recently elucidated crystal structures of potassium channels reveal similar structural motifs at their pore-forming regions, suggesting that they share a common gating mechanism. The validity of this hypothesis is explored by analyzing the collective dynamics of five known K(+) channel structures. Normal-mode analysis using the Gaussian network model strikingly reveals that all five structures display the same intrinsic motions at their pore-forming region despite the differences in their sequences, structures, and activation mechanisms. Superposition of the most cooperative mode profiles shows that the identified common mechanism is a global corkscrew-like counterrotation of the extracellular and cytoplasmic (CP) regions, leading to the opening of the CP end of the pore. A second cooperative mode shared by all five K(+) channels is the extension of the extracellular and/or CP ends via alternating anticorrelated fluctuations of pairs of diagonally opposite monomers. Residues acting as hinges/anchors in both modes are highly conserved across the members of the family of K(+) channel proteins, consistent with their presently disclosed critical mechanical role in pore gating.

FIGURE 1

Sequence and structure of the pore region of five structurally known potassium channels. (a) Alignment of the pore-region sequences. See Table 1 for the nomenclature and source. The regions corresponding to the helices TM1 and TM2 and the P-loop are indicated by yellow, green, and red arrows, respectively. The alignment was performed using ClustalW (63) Fully or highly conserved regions are shown in black, moderately conserved in gray. Two regions of interest are the signature motif GYG at the selectivity filter, and the conserved G on TM2, both enclosed in magenta boxes (see also Fig. 2 a). (b) Structural comparison of the pore-forming regions of the K⁺ channels aligned in a. These are all tetrameric structures, the monomers of which contain either two TM helices (KcsA, MthK, and KirBac), colored yellow (TM1) and green (TM2), or six TM helices (KvAP and Shaker), denoted as S1–S6. Only the pore-forming helices, S5 and S6, equivalent to TM1 and TM2, are displayed here, along with the P-helix/loops region, colored red.

FIGURE 2

Structure and dynamics of KcsA. (a) Ribbon diagram of the KcsA, based on the PDB structure deposited by Doyle et al. (3). Each monomer contains two TM helices, TM1 (outer; yellow) and TM2 (inner; green). The flow of K⁺ ions across the selectivity filter based on MD simulations (15) is schematically shown. Other regions of functional importance are the gate near the intracellular region and the turret at the extracellular surface. The short black arrows indicate the movements suggested by spin labeling experiments (13) for gate opening. The figure was made using Molscript (64). (b) Equilibrium fluctuations as a function of residue index. Left and right ordinates refer to the B-factors and mean-square fluctuations, respectively, of individual residues, which are related as B_i = 8π²〈(ΔR_i)²〉/3, where i is the residue index (abscissa). The three curves describe the results obtained from GNM analysis (red), MD simulations (black), and x-ray crystallography (blue). The correlation coefficients between experimental data and theoretical results are 0.94 (GNM) and 0.83 (MD), and that between the two theoretical results is 0.93.

FIGURE 3

Residue mobilities induced by two dominant mechanisms of global motion. The panels describe the square displacements of residues driven by the two most cooperative (lowest-frequency) motions predicted by the GNM, referred to as global motions of type I and II, for all five structures examined (see Fig. 1 b). The left panels display the results for motion I, for the entire tetramer. This motion results from the combination of two degenerate modes that each activate the diagonally positioned pairs of monomers (see Fig. 3). The right panels display the mobilities induced by both modes I (black) and II (red). Results are displayed for monomers only, as they are repeated across all four monomers. Minima indicated by blue “x” and red dot symbols on the abscissa coincide with the signature sequence GYG, and the conserved Gly residues on the TM2 helices, respectively (Fig. 1 a). Those indicated by solid triangles on the right refer to small hydrophobic residues at central positions on TM1 helices, which closely interact with the conserved Gly on TM2 helices. These three sets define the key sites that act as hinges/anchors in coordinating the two global mechanisms.

FIGURE 4

Mobilities in global modes shown by color-coded diagrams for KcsA, KirBac, and Shaker. The slowest global mode (mode type I) is twofold degenerate. The corresponding ribbon diagrams are shown on the left and middle columns. The second-lowest-frequency mode (mode type II) is illustrated in the right column. Color code is red, orange, green, cyan, and blue in order of decreasing mobility. Note that mode type I involves the pairwise motions of oppositely positioned monomers, whereas mode type II is cylindrically symmetric.

FIGURE 5

Master curves for global dynamics of potassium channels. (a) Superposition of the most cooperative global mode shape (type I) for KcsA (red), KirBac (green), KvAP (blue), Shaker (magenta), and MthK (black), plotted for one monomer. The abscissa refers to the residue indices of MthK. (Inset) Ribbon diagram of KcsA, with two monomers removed for clarity, highlighting the selectivity-filter residues GYG, in space-filling representation. (b) Superposition of the global mode shape (type II) for the same set of proteins. The abscissa refers to the residue indices of MthK. (Inset) Ribbon diagram of KcsA, with two subunits removed for clarity, highlighting the conserved glycine on TM2 in space-filling representation.

FIGURE 6

Dynamic equilibrium between fluctuating conformations. Ribbon diagrams represent different deformed models for KcsA (a and b) and Shaker (c), generated using Eq. 6 for mode types I and II. (a) Side view of deformed model for type I mode for s_I = −75 (top) and s_I = −100 (bottom). (b) Top view of deformed models for mode type I obtained using s_I = −75 (top right), s_I = +75 (bottom left), and for mode type II, s_II = −100 (top left), s_II = 100 (bottom right). The figure in the middle is that of the crystal structure. (c) Counterpart of b for Shaker.

FIGURE 7

(a) Pore-radius profiles as a function of the position along the cylindrical (z) axis. The radii are computed for the crystal structure (black) and the deformed conformations at s_II = −200 (red solid line) and s_II = +200 (red dashed line). (b) Solid-sphere representation of the inner surface of the channel at the pore region for the crystal structure (left), and for the model of the open form (right) at s_II = −200, with the corresponding backbone structures superimposed onto the pore surface. The color code for the solid-sphere representation is: red, pore radius <1.15 Å; green, 1.15 Å < radius < 2.30 Å; and blue, radius >2.30 Å. The pore-radius profiles were generated using HOLE (65). In the inset of a is the backbone of the crystal structure (blue) superimposed onto the model of the open form (red). Two monomers have been deleted for clarity.

FIGURE 8

Cartoon representation of the TM2 helices of the deformed models of KcsA, KirBac, and Shaker, at different values of scaling parameter (Eq. 6). (a, top row) Cartoon representation of closed and open models for KcsA, proposed as a model for molecular mechanism of gating, based on experimental data (Fig. 7 in Perozo et al. (13)). (a, middle row) TM2 helices in the original structure (s_II = 0) and in the conformation induced by mode II (s_II = −100) for KcsA. (a, bottom row) TM2 helices of Shaker in the PDB structure (s_II = 0) and the predicted structure deformed along mode II (s_II = −55). (b, top row) Cartoon representation of KcsA (red) and MthK (green) TM structures, illustrating the model proposed by Perozo and co-workers for a “hinge-gating” mechanism at the intracellular gate. G⁸³ acts as a hinge (Fig. 1 in Perozo (14)). (b, middle row) KcsA TM2 helices at s_II = −150, from the top (left) and side (right). Highlighted residue at the kink is T¹⁰⁷. (b, bottom row) KirBac TM2 helices at s_II = −73, from the top (left) and side (right). Conserved glycine (G¹³⁴) emerging as kink residue is highlighted.