Structural basis of lipid-driven conformational transitions in the KvAP voltage-sensing domain

Abstract

Voltage-gated ion channels respond to transmembrane electric fields through reorientations of the positively charged S4 helix within the voltage-sensing domain (VSD). Despite a wealth of structural and functional data, the details of this conformational change remain controversial. Recent electrophysiological evidence showed that equilibrium between the resting ('down') and activated ('up') conformations of the KvAP VSD from Aeropyrum pernix can be biased through reconstitution in lipids with or without phosphate groups. We investigated the structural transition between these functional states, using site-directed spin-labeling and EPR spectroscopic methods. Solvent accessibility and interhelical distance determinations suggest that KvAP gates through S4 movements involving an ∼3-Å upward tilt and simultaneous ∼2-Å axial shift. This motion leads to large accessibly changes in the intracellular water-filled crevice and supports a new model of gating that combines structural rearrangements and electric-field remodeling.

Figure 1

Functional and conformational changes induced by lipids. (a) Voltage dependence of KvAP was dramatically right shifted in the presence of non-phosphate lipids: 80% DOTAP with 20% POPE:POPG (black, V_h=73 mV, z=1.5) comparing to the phosphate lipids: 100% POPE:POPG = 3:1 (red, V_h=−42 mV, z=3.1). According to the simulated G-V curves, VSD of KvAP populates predominantly at activated (“Up”) state in phosphate lipids and at resting (“Down”) state in non-phosphate lipids at 0 mV, in the absence of asymmetric voltage under biochemical conditions. (b) Spin label was introduced to positions 16-147 on KvAP VSD one at a time for EPR spectroscopy studies in both phosphate lipid POPC:POPG=3:1 (PCPG) and non-phosphate lipid DOTAP. (c) Mobility ΔH_o⁻¹, oxygen accessibility ∏O₂ and NiEDDA accessibility ∏NiEdda of KvAP VSD in PCPG (red) and DOTAP (black). 20 selected positions were repeated (n=3-5) and the standard deviation were shown in grey error bar. The grey regions represent the four putative transmembrane segments (S1, S2, S3 and S4) from the crystal structure. To facilitate comparison between the two conditions, we colored the area between the two data sets for both O₂ and NiEdda accessibility profiles to highlight the degree and direction of the accessibility changes. Using the PCPG data (Up conformation) as reference, accessibility increases in the DOTAP-reconstituted sensor are colored in yellow, whereas accessibility reductions are shown as light blue areas. ∏NiEdda of S4 has a decreasing trend at the extracellular side and increasing trend at the intracellular side emphasized by the yellow arrows.

Figure 2

The lipid dependent conformation change. (a) Changes in water penetration leads to a refocusing of the transmembrane electric field. NiEdda data mapped onto the crystal structure of the isolated KvAP-VSD for DOTAP (left) and PCPG (center). The color spectrum is a linear scale between white (lowest NiEdda accessibility) and blue (highest). The red spheres depict the lowest two putative gating charges R133 (R6) and K136 (K7) in KvAP. Right, theoretical depiction of the voltage drop along KvAP-VSD after reconstitution in DOTAP (red) or PCPG (black). (b) The delta (DOTAP-PCPG) of ΔH_o⁻¹, ∏O₂ and ∏NiEdda were plotted for the S4 region. There is clearly a tilt trend of a ∏NiEdda shown as a decrease at the top of S4 and increase of the bottom. (c) The delta of ΔH_o⁻¹, ∏O₂ and ∏NiEdda were mapped onto the crystal structure of KvAP VSD. The bottom crevice has dramatic increase of ∏NiEdda accessibility accompanied by the slight increase from top crevice and decrease at S3-S4 loop region. There are obvious increases of ∏O₂ at top of S4 (yellow arrow on Δ∏O₂ map) suggesting the region getting closer into lipids.

Figure 3

Quantitation of the conformation change by DEER distance measurement in lipids. (a) The scheme of the bi-functional spin label. Each distance between a pair of spin labels requires the introduction of four designed cysteine residues. (b) Representative raw DEER data (left) for a pair of distance measurement between 57/61 (bi-functional spin label attaching to position 57 and 61) and 118/121, and the distance distribution from Tikhonov regularization (right). The changes are small but confidently differentiable. (c) The absolute distances of 10 pairs in PCPG disagree with the crystal structure in a systematic way. It indicates that the crystal structure is over tilted 5~8 Å more than in PCPG. (d) The delta distances (DOTAP-PCPG) suggest a slight tilt and down movement of S4 at 2~3 Å. The distance change from PCPG to DOTAP is consistent in the direction of tilt of the S4.

Figure 4

The lipid induced conformation change on KvAP VSD is titratable. (a) Spin labeled KvAP VSD proteins at four individual positions (112, 114, 140 and 145) were titrated with increments in the percentage of DOTAP. Residues 112 and 114 are on the extracellular side of S4 and residues 140 and 145 are on the intracellular side of S4. (b) Continuous wave EPR spectra of residues 112 and 145 upon DOTAP titration. The spectra were normalized by number of spins. As the percentage of DOTAP increases, the amplitudes are decreasing for residue 112 and increasing for residue 140. (c) The solvent accessibility (∏NiEdda) of S4 are decreasing for extracellular side (112, 114) and increasing for the intracellular side (140, 145). (d) The mobility ΔH_o⁻¹ change of extracellular side of S4 (112, 114), intracellular side of S4 (140, 145) and the pore (226, 228). The change on the intracellular side of S4 and the pore overlapped in the range of 40-100% DOTAP.

Figure 5

The Tilt-Shift model. (a) The distance change from Up state to Down state within KvAP VSD. (left) Up state VSD model (grey) was generated by MD simulation. The three Down states based on helix-screw (magenta), Paddle model (orange) and Tilt-Shift (blue) models. (right) The expected distance changes according to three models were plotted against experimental differences. The grey region indicated the uncertainty of measurement. Neither the helix-screw or the Paddle model is compatible with the experimental data, and only the Tilt-Shift model offer plausible explanation. (b) The alternative explanation to the existing data. (left) All the established state dependent accessibilities on KvAP focused on the region of 121-125. A Paddle model proposed a 15-20 Å downward movement of S4 to account for the their accessibility from the bottom. (c) Δ∏NiEdda was mapped on to the crystal structure. It shows the water penetration deep into the bottom crevice, where the region 121-125 is only ~ 5 Å above. Our observed ~ 3 Å Tilt-Shift of S4 should be sufficient to account for their accessibility from the bottom crevice. (d) Cartoon representation of the Tilt-Shift model on KvAP VSD. The ~25° tilt and ~2 Å down shift of the S4 could generate a 3~4 Å movement of the S4-S5 linker which seems to sufficient to open the pore and increases permeation of potassium current.