General rules for the arrangements and gating motions of pore-lining helices in homomeric ion channels

Abstract

The pore-lining helix (PLH) bundles are central to the function of all ion channels, as their conformational rearrangements dictate channel gating. Here we explore all plausible oligomeric arrangements of the PLH bundles within homomeric ion channels by building models using generic restraints. In particular, the distance between two neighbouring PLHs was bounded both below and above in order to avoid steric clash and allow proper packing. The resulting models provide a theoretical representation of the accessible space for oligomeric arrangements. While the represented space is confined, it encompasses nearly all the ion channel PLH bundles for which the structures are currently known. For a multitude of channels, gating models suggested by paths within the confined accessible space are in qualitative agreement with those established in previous structural and computational studies.

Figure 1. Pore-lining helix bundle, modelled as consisting of ideal helices obeying rotational symmetry.

(a) Top view of KcsA (Protein Data Bank entry 1BL8), with the pore-lining helix bundle highlighted in green. (b) Side view of an idealized helix bundle. The rotational symmetry axis, pointing towards the N termini of the helices, is defined as the z axis; the centres of the protomers lie on the xy plane, with the y axis pointing from the centre of the first protomer (shown in yellow) to the z axis. The helical axis of the first protomer crosses the xy plane at the point (0, −r, 0) and has polar angle θ and azimuthal angle φ. (c) Top view of the idealized bundle. (d) The contact distance d, defined as the closest distance between backbone N, C_α, C and O atoms of two neighbouring helices.

Figure 2. Distribution of the C_α RMSDs between the pore-lining helix bundles in 39 channel structures and their idealized substitutes.

Superposition to idealized substitutes (orange) is shown for Kir3.1 (PDB 2QKS), KvAP (PDB 2A0L) and ASIC1a (PDB 4FZ1) (blue), with low (0.6 Å), medium (1.4 Å), and high (2.0 Å) RMSDs, respectively.

Figure 3. Exhaustive exploration of the conformational space accessible to pore-lining helix bundles.

(a) A slice (at r=9 Å) through the conformational space for tetrameric bundles. Each starting model is represented by a dot on the θ–φ plane. Models that were eliminated by d<3 Å (steric clash), by d>7 Å (poor packing) and by clash between C_β atoms as well as backbone atoms of non-nearest neighbouring helices are coloured in black, grey and teal, respectively; the surviving models are coloured in red. (b) Top row: tilting of the helical axis away from (or towards) the z axis, corresponding to negative (or positive) φ, results in the larger (or smaller) pore opening towards the N termini located on the red dashed circle. Middle row: bundles with right-handed and left-handed twists have positive and negative cosφ, respectively. Bottom row: helical bundles with four distinct gross appearances, one from each branch of the W-shaped accessible region. (c) Percentages of eliminated (either by steric clash or poor packing) and surviving models in different oligomeric states.

Figure 4. The W-shaped accessible region in the r–θ–φ space.

(a) The accessible region for tetrameric models, embedding the conformations of 23 actual PLH bundles. The r values of the plausible models are displayed according to the colour scale at the top. Each black dot represents an actual PLH bundle. (b) Corresponding results for pentamers. In a,b, all the black dots are within the respective W shapes (that is, each black dot is within a voxel defined by eight plausible models as vertices that are separated by the smallest increments in (r, θ, φ)). (c,d) The conformations of the actual tetrameric and pentameric PLH bundles displayed on the θ–φ plane. In each plot a yellow oval encircles two channels that are either not or only distantly related but have similar conformations for their PLH bundles.

Figure 5. Similar conformations for the pore-lining helix bundles of channels that are either not or only distantly related.

(a) Superposition of the TM2 helices of desensitized ASIC1a in blue (PDB 3HGC) and apo P2X4 receptor in red (PDB 4DW0). Segments in grey were excluded from superposition. (b) Superposition of the TM2 helices of NaK channel in blue (PDB 2AHY) and TM3 helices of AMPA-subtype iGluR in red (PDB 3KG2), both in the closed state. (c) Superposition of the TM2 helices of locally closed GLIC in blue (PDB 3TLS) and the transmembrane helices of T-state phospholamban in red (PDB 2KYV).

Figure 6. N-dependent distributions of the r, θ and φ coordinates for the plausible models and for the pore-lining helices of the 39 channel structures.

Distributions of r, θ and φ are displayed in a–c, respectively. Results are displayed as curves for the plausible models and as bars for the channel structures.

Figure 7. Minimum pore radii of the plausible models and gating models of three channels suggested by paths of pore expansion.

(a) The R₀ values of the plausible trimeric models are displayed according to the colour scale at the top. A path connecting the closed P2X4 receptor (PDB 4DW0) to an open model represents a hypothetical gating mechanism. The first and second branches are plotted with φ shifted by 360° so that the path from the fourth branch to the first branch is connected. The open-state crystal structure (PDB 4DW1) is shown as a grey dot. (b,c) Corresponding results for the tetrameric models and KcsA (path connecting the closed (PDB 1BL8), partially open (PDB 3FB6) and open (PDB 3F5W) states), and for the pentameric models and MscL (path starting from the closed state (PDB 2OAR)), without the shift in φ. (d–f) Illustrations of the gating models for the P2X4 receptor, KcsA and MscL, using conformations shown as black dots in a–c, respectively. Bundles in blue, grey and red represent closed, intermediate and open conformations, respectively, shown in top view (top panels) and side view (bottom panels).