The voltage-gated sodium channel pore exhibits conformational flexibility during slow inactivation

Figures

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  Figure 1.. NaVSp1-P pore-domain adopts a conductive conformation. (A) A schematic representation of the full-length NaVSp1 with the VSD and the PD marked. Only two subunits are shown for clarity. An exemplar recording of NaVSp1 whole-cell Na+ currents. The activation protocol consisted of 500-ms depolarization from −80 to +40 mV in 5-mV increments from a holding potential of −120 mV with a sweep-to-sweep interval of 4 s. The inset shows a typical response to 0-mV depolarization pulse, where the steady-state open probability is less than 5% of the peak value (left). NaVSp1 currents elicited by pulse-protocol (shown above). At the end of a 0-mV prepulse, the test pulse to 10 mV evokes minimal inward current, indicating that the channels are inactivated at the end of the prepulse (right). (B) Single-channel recordings of NaVSp1-P reconstituted in liposomes (a schematic of NaVSp1-P shown above) show functionally active channels that stochastically open and close. The trace within the gray box is shown in an expanded scale. C and O denote the closed and open states. The dotted line marks the zero-current level. (C) Two diagonal subunits of closed-gate NaVAb crystal structure (PDB ID 3RVY) with…

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  Figure 2.. Characterization of individual cysteine mutants. (A) Elution profiles from size exclusion chromatography for WT and representative spin-labeled cysteine mutants before reconstitution. (B) Whole-cell current traces for representative NaVSp1 cysteine mutants recorded from HEK-293 cells in response to an activation protocol consisting of 500-ms depolarizations from −80 to +100 mV in 5-mV increments from a holding potential of −120 mV with a sweep-to-sweep interval of 4 s. The bottom panel is the voltage dependence of activation for WT and cysteine mutants. The V1/2 of activation is WT (28.9 ± 0.5 mV, n = 3); P156C (26.1 ± 0.5 mV, n = 3); D157C (25.5 ± 0.7 mV, n = 3); F170C (21.2 ± 1.1 mV, n = 4); V186C (29.0 ± 0.4 mV, n = 4); and V189C (29.0 ± 0.4 mV, n = 3). The vertical lines show SD from “n” individual measurements.

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  Figure 3.. Conformational differences in the selectivity filter region of NaVSp1 and NaVSp1-P. Spin-normalized CW-EPR spectra for positions along the turret loop, P1 helix, and P2 helix in NaVSp1 (red) and NaVSp1-P (black). These regions are color-coded on the NaVAb structure (PDB ID 3RVY). Dotted lines marked as “i” and “m” represent the immobile and mobile components of the spectra, respectively. The two components may arise from two different rotameric orientations of the spin labels and/or from two conformational states of the protein in equilibrium. Arrows highlight positions with the most prominent differences in line shapes in the two channels.

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  Figure 4.. A close-up view of the intersubunit cavity in the NaVAb structure. Residues that show a prominent increase in mobility in NaVSp1 in comparison to NaVSp1-P are shown in red, and those that show a decrease in this parameter are represented in blue. The residues with higher mobility values are found to line the intersubunit cavity, which is at the interface of P1 and P2 helices from two adjacent subunits.

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  Figure 5.. EPR environmental parameters reveal conformational differences in the membrane-embedded NaVSp1 and NaVSp1-P. (A) A plot of residue environmental parameters for NaVSp1-P (shown in gray) and NaVSp1 (shown in color). ΔHo−1 parameter (top); O2 accessibility, ΠO2 (middle); water accessibility, ΠNiEDDA (bottom). The EPR parameters for continuous positions are linked by solid lines. Residues that were not studied (because of sub-optimal expression/oligomeric stability) appear as gaps. Positions revealing prominent changes are highlighted within blue circles. (B) Difference in ΠO2 values between the NaVSp1 and NaVSp1-P are mapped on the NaVAb structure (PDB ID 3RVY) and color-coded, red denoting an increase and blue representing a decrease in the environmental parameter. (C) A plot of the average lipid accessibility (ΠO2) as a function of the average mobility parameter ΔHo−1 for the turret loop (yellow), P1 helix (green), and P2 helix (magenta). The light gray boxes represent NaVSp1-P, and the black boxes denote NaVSp1. The dotted lines connecting the light and black boxes for each region signify the magnitude of difference between the EPR environmental parameters in the two channels.

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  Figure 6.. Extent of conformational change measured by DEER spectroscopy. NaVAb structure showing the positions investigated by DEER (PDB ID 4EKW). Fit to the background subtracted DEER echo intensity is plotted against evolution time. The corresponding interspin distance distribution (right) for the NaVSp1-P (black) and NaVSp1 (red) for different spin-labeled positions. The two expected distances (Cβ–Cβ) from the adjacent and nonadjacent subunits in NaVAb crystal structure are shown in black dotted lines. The shorter distance is from the adjacent subunits and the longer from the diagonal subunits. The dotted gray lines are distance distributions from rotamer simulation for respective positions on PDB ID 4EKW (potentially slow-inactivated NaVAb conformation) using MMM. The arrows highlight the direction of change. Asterisk denotes distance peaks that may arise from intermolecular spin interactions between individual tetramers.

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  Figure 7.. Spectral changes at the SF region underlying transitions between the putative resting/conductive and slow-inactivated states. CW spectra for NaVSp1 at representative positions in the P2 helix (positions marked on the NaVAb structure) reconstituted in asolectin (red) and DOTAP (cyan) and compared with NaVSp1-P reconstituted in asolectin (black). A similarity of NaVSp1-DOTAP and NaVSp1-P–asolectin spectra is highlighted in the gray box.

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  Figure 8.. Electrophysiological characterization of slow inactivation in a mutant channel NaVSp1-170C. (A) Use-dependent development of slow inactivation: depolarizations from a holding potential of −120 mV to 20 mV were applied at 0.2 Hz (left, for 200 ms in duration) and 1 Hz (right, for 150 ms in duration) frequencies, and the normalized peak current for each pulse was plotted as a function of the pulse number for NaVSp1-WT (gray squares) and NaVSp1-F170C (blue squares). The error bars denote SDs from n = 4 for NaVSp1-WT and n = 5 for NaVSp1-F170C. (B) Voltage dependence of inactivation: peak currents were measured during test pulses to 20 mV after a 2-s inactivating pulse to the indicated potentials. Values from individual experiments were normalized to the maximum test pulse currents. Inactivation curves were fit with a Boltzmann equation, 1/1+exp[(V−Vh)/kh], where Vh is the half-inactivation voltage and kh is the slope factor. For NaVSp1-WT, mean Vh was −30.3 ± 1.7 mV (n = 4); for NaVSp1-F170C (n = 4), Vh was −35.7 ± 1.3 mV. (C) Time constants of inactivation versus voltage curves for NaVSp1-WT (gray squares, n = 4) and NaVSp1-F170C (blue squares, n = 4) obtained by fitting a single…

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  Figure 9.. An EPR-based model for the mechanism of slow inactivation at the SF region. A cartoon representation of putative changes in the P1 and P2 helices during slow inactivation. These positional differences are likely to change intersubunit cavity volumes and hence, the accessibility of lipids and lipophilic molecules into these vestibules. The region in the inset is shown from the top (extracellular end).