The activated state of a sodium channel voltage sensor in a membrane environment

Abstract

Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na(+) and Ca(2+) channel families. Here, we report the spectroscopic structural characterization of the isolated Voltage-Sensor Domain (VSD) of the prokaryotic Na(+) channel NaChBac in a lipid bilayer. Site-directed spin-labeling and EPR spectroscopy were carried out for 118 mutants covering all of the VSD. EPR environmental data were used to unambiguously assign the secondary structure elements, define membrane insertion limits, and evaluate the activated conformation of the isolated-VSD in the membrane using restrain-driven molecular dynamics simulations. The overall three-dimensional fold of the NaChBac-VSD closely mirrors those seen in KvAP, Kv1.2, Kv1.2-2.1 chimera, and MlotiK1. However, in comparison to the membrane-embedded KvAP-VSD, the structural dynamics of the NaChBac-VSD reveals a much tighter helix packing, with subtle differences in the local environment of the gating charges and their interaction with the rest of the protein. Using cell complementation assays we show that the NaChBac-VSD can provide a conduit to the transport of ions in the resting or "down" conformation, a feature consistent with our EPR water accessibility measurements in the activated or "up" conformation. These results suggest that the overall architecture of VSD's is remarkably conserved among K(+) and Na(+) channels and that pathways for gating-pore currents may be intrinsic to most voltage-sensors. Cell complementation assays also provide information about the putative location of the gating charges in the "down/resting" state and hence a glimpse of the extent of conformational changes during activation.

Fig. 1.

EPR based structural analysis of the isolated-VSD. (A) Mobility (black), O₂ accessibility ΠO₂ (red) and NiEdda accessibility ΠNiEdda (blue). The gray regions represent the putative TM segments from the hydropathy plot. Green asterisks denote highly conserved residues with VSDs. Bent arrows point to the increase in ΠO₂ towards the center of the bilayer in S1 and S2 and straight arrows indicate a break in helix periodicity in S3 and S4. (B) Helical wheel representation of ΠO₂ superimposed in a polar coordinate representation. The relative orientation of the helices can be predicated based on the direction of the resultant vector, which points towards the lipid facing phase of the helix. The shaded area within the dashed lines highlights the degree of eccentricity for the complete set of accessibility data relative to the maximal accessibility vector.

Fig. 2.

Environment of the S4 segment (A) Profile of changes in the ΠO₂ parameters reflects a break in periodicity. Fourier transform power spectra of ΠO₂ shows that the peak angular frequency occurs at ∼99.8° for the upper-half of S4 corresponding to an α-helix, while it is at ∼118.1° for the lower-half suggesting a 3₁₀ helical structure. (B) An overlap of representative X-band CW-EPR spectra of spin-labeled mutants at corresponding arginines in S4 for the VSD from NaChBac (red) and KvAP (black). Blue line shows the location of immobile component of the spectra (Left). Position of arginine side-chains as seen in the crystal structure of the isolated-VSD of KvAP (center). An overlap of EPR spectra of the corresponding positions based on the alignment of NaChBac and KvAP VSDs (Right). The shaded boxes highlight positions that show maximal change in line shape.

Fig. 3.

Aqueous crevices within the VSD. (A) ΠNiEdda values for the residues comprising the S3–S4 segment in the isolated-VSD. (B) ΠNiEdda values mapped on the S3 segment of the NaChBac-VSD model to show the depth of water accessible areas (blue arrows) within the transmembrane region of the protein. The dotted line denotes the putative limits of the membrane. (C) LB2003 complementation assay. Mutant strain LB2003 transformed with Gly mutants at R1 (green triangles), R2 (blue inverted triangles), and R3 (cyan diamonds), the negative control is closed wt-KcsA (black squares) and the positive control is the C-terminal truncated KcsA (Δ120) (red circles).

Fig. 4.

A model of the activated state of the NaCh-VSD obtained from EPR-based refinement. Mobility parameter (), O₂ accessibility parameter (ΠO₂) and NiEdda accessibility parameters (ΠNiEdda ) are mapped on to a surface representation of the model.

Fig. 5.

All-atom MD simulations of the EPR based model of NaChbac-VSD (A) A snapshot of the system embedded in membrane and water. Red and white surface was rendered for water molecules. R2, R3, and R4 side-chains are shown in stick representation. (B) EPR based model of NaChBac-VSD showing the location of gating arginines. (C) Density profiles for R3, D60, R4, lipid, and water during the last 5 ns of the free MD simulation. For clarification, 20 times magnification of the graphs has been applied for the three residues. (D) The putative “up” and “down” states of the NaChBac-VSD