A Mechanistic Reinterpretation of Fast Inactivation in Voltage-Gated Na + Channels

Abstract

Fast Inactivation in voltage-gated Na ⁺ channels plays essential roles in numerous physiological functions. The canonical hinged-lid model has long predicted that a hydrophobic motif in the DIII-DIV linker (IFM) acts as the gating particle that occludes the permeation pathway during fast inactivation. However, the fact that the IFM motif is located far from the pore in recent high-resolution structures of Nav ⁺ channels contradicts this status quo model. The precise molecular determinants of fast inactivation gate once again, become an open question. Here, we provide a mechanistic reinterpretation of fast inactivation based on ionic and gating current data. In Nav1.4 the actual inactivation gate is comprised of two hydrophobic rings at the bottom of S6. These function in series and closing once the IFM motif binds. Reducing the volume of the sidechain in both rings led to a partially conductive inactivated state. Our experiments also point to a previously overlooked coupling pathway between the bottom of S6 and the selectivity filter.

Figure 1:

Pore radius profile of NavPas and Nav1.7. A) Structure of NavPas (PDB:6A95 - (30)). B) Structure of Nav1.7 M11 (PDB:7XVF - (28). The IFM motif is highlighted in red. The pore radius calculated using HOLE software (31). NavPas and Nav1.7 structures show similar pore profile at the bottom of S6, where two layers of hydrophobic barrier are identified (shadowed regions). Identified top (extracellular view) and bottom (intracellular view) hydrophobic layers in NavPas and Nav1.7 are shown. In NavPas, the top layer is formed by residues L412 (DI), I737 (DII), V1108 (DIII), A1407 (DIV) and the bottom layer by V416 (DI), L741 (DII), I1112 (DIII), L1411 (DIV). In Nav1.7, the top layer is formed by residues L390 (DI), L964 (DII), I1453 (DIII), I1756 (DIV) and the bottom layer by A402 (DI), L968 (DII), I1457 (DIII), L1760 (DIV). C) Sequence alignments of S6 region among rNav1.4, Nav1.7 and NavPa. The residues identified to form the narrowest part of the channel were highlighted in light blue for NavPas and dark blue for Nav1.7, and they are colored in red for rNav1.4.

Figure 2:

Double alanine mutation in DIII S6 relieves fast inactivation on rNav1.4. A) Top: sequence alignment of DIII S6 showing identified residues (I1284 and I1288, highlighted in yellow). Bottom: Representative ionic traces for the wild-type (WT), single (I1284A and I1288A) and double alanine (I1284A_I1288A, named DIIIAA) mutations of the identified residues on rNav1.4. The ionic conditions used was 57.5 Na⁺ outside and 12 Na⁺ inside. Inset shows the voltage protocol. B) Ratio of steady state current (I_ss, ●) at the end of depolarization of 30ms over the peak current (I_peak, ■) taken at +60mV. C) Voltage-dependence of inactivation (h-infinity curve) for WT (black), I1284A (green), I1288A (blue) and DIIIAA (red). The curves were made by plotting the normalized peak ionic current (Inorm) at the test pulse (60mV) after 50ms conditioning prepulse (voltage protocol in inset). The lines represent the fit to a two-state model (Equation 1). Data are shown as Mean ± SEM. Number of cells tested are in table I.

Figure 3:

Evidence of a conductive “inactivated” state. Representative traces of gating current used for charge immobilization measurements for WT (A) and DIIIAA (B), bottom inset in (A) shows the voltage protocol. Black square top inset shows the comparison of the normalized gating charge for a 0.5 ms and 23 ms depolarization pulse. C) Fraction of immobilized charge vs depolarization time for WT (black) and DIIIAA mutant (red). The inset illustrates the method used for determination of the off-gating components using exponential fitting (Detailed in methods). D) Fast Inactivation time constant for WT (black) and DIIIAA (red). Fast inactivation kinetics were characterized by fitting a single exponential function for WT and two-exponential function for DIIIAA. Inset shows representative DIIIAA current traces in response to a voltage protocol shown in Figure2A. E) DIIIAA currents in response to depolarized voltage step to +60mV with variable duration followed by a hyperpolarized voltage step to −90mV. The inset shows the normalized tail currents at 3 depolarization times. F) Weighted tail currents time constant vs depolarization time for DIIIAA mutant (Details in methods). Data are shown as Mean ± SEM.

Figure 4:

Time dependent selectivity changes in DIIIAA. DIIIAA depolarization activated currents at different voltages (range shown on top) using 57.5 mM external Na⁺ (A), 90 mM external Na⁺ (B) or 120 external K⁺ (C), on the right I-V curves for the peak (red) and steady state (blue) current. The reversal potential for each component is denoted with a corresponding dashed line. D) Example of the changes in direction for the tail currents. E) Instantaneous I-V curves after a 0.6 (red) 1 (blue) 5 (green) and 20 (black) milliseconds depolarization times. The solid lines represent a linear fit used to obtain the reversal potential (indicated by vertical dashed lines). F) Relative Na/K permeability vs time for DIIIAA under different ionic conditions (red, 57.5 mM Na external and green, 90 mM Na external concentration. Internal solution 120 mM K. Dashed line shows the WT permeability and solid lines represent an exponential fit to obtain the time constant. G) Fast time constants for the change in selectivity (DIVAA Tau_perm) and fast inactivation (DIIIAA Tau_FI).

Figure 5:

Preventing IFM binding avoids the DIIIAA phenotypes. A) G-V curves for WT (black dashed line), I1284A (green), I1288A (blue) and DIIIAA (red). GV curves were calculated from the peak for WT, I1284A and I1288A whereas for DIIIAA it was calculated from the steady state currents. B) Representative gating current traces for WT and DIIIAA. Inset is the voltage protocol. C) Q-V curves for WT (black) and DIIIAA (red). The dashed lines indicate the corresponding G-V curves. D) Representative ionic current traces for IQM and IQM_DIIIAA. E) G-V curves for IQM (magenta) and IQM_DIIIAA (orange). Dashed lines show the WT and DIIIAA for comparison. F) IQM_DIIIAA IV curves for the peak (red) and steady state (blue) current. The reversal potential for each component is denoted with a corresponding dashed line. G) IQM_DIIIAA currents in response to depolarized voltage step to +60mV with variable duration followed by a hyperpolarized voltage step to −90mV. The inset shows the normalized tail currents at 3 depolarization times. H) Weighted tail currents time constant vs depolarization time for IQM_DIIIAA (red points) compared to DIIIAA (dashed line).

Figure 6:

Alanine mutations in DIV also produced a leaky inactivated state. A) Top: sequencing alignment of DIV S6 showing identified residues (I1587 and L1591, highlighted in yellow). Bottom: Representative ionic traces for single (I1587A and L1591A) and double alanine (I1587A_L1591A – DIVAA) mutations. The ionic conditions used was 57.5 Na⁺ outside and 12 Na⁺ inside. B) Ratio of steady state current (I_ss) at the end of depolarization over the peak current (I_peak) taken at +60mV. C) Voltage-dependence of inactivation (h-infinity curve) for WT (black), I1587A (green), L1591A (blue) and DIVAA (red). D) Representative gating currents traces for charge immobilization measurements for DIVAA. E) Fraction of immobilized charge vs depolarization time for WT (black) and DIVAA mutant (red). F) IV curves for the peak (red) and steady state (blue) current for DIVAA. The reversal potential for each component is denoted with a corresponding dashed line. F) Relative Na/K permeability vs time for DIVAA (57.5 mM Na external and 120 mM K internal). Dashed grey and blue lines show the WT and DIIIIAA permeability, respectively. Solid line represents an exponential fit to obtain the time constant. G) Fast time constants for the change in selectivity (DIVAA Tau_perm) and fast inactivation (DIVAA Tau_FI). Data are shown as Mean ± SEM.

Figure 7:

Quadruple alanine mutation of the identified residues in DIII & DIV S6. A) Representative current traces elicited at different voltages (−10 to 40 mV) for the quadruple alanine mutation (I1284A_I1288A_I1587A_L1591A, DIIIAA_IVAA) using 57.5 mM external Na and 120 internal K. (B) IV curves for the peak (red) and steady state (blue) current. The reversal potential for each component is denoted with a corresponding dashed line. C) Relative Na/K permeability vs time for DIVAA (57.5 mM Na external and 120 mM K internal). Dashed grey, blue and red lines show the WT, DIIIIAA and DIVAA permeability, respectively. Solid line represents an exponential fit to obtain the time constant. D) h-infinity curve for DIII_IVAA, DIIIAA and DIVAA. Significant fast inactivation was removed in DIII_DIVAA.

Figure 8:

Effects of alanine mutations in DI and DII S6. A) Top: sequencing alignment of DI S6 showing identified residues (L437 and A441, highlighted in yellow). Bottom: Representative ionic traces for L437A. The ionic conditions used was 57.5 Na⁺ outside and 12 Na⁺ inside. B) G-V curves for WT (black dashed line) and L437A (red). GV curves were calculated from the peak. C) H-infinity curve for WT (black) and L437A (red). D) Top: sequencing alignment of DII S6 showing identified residues (L792 and L796, highlighted in yellow). Bottom: Representative current traces for L792A_L796A (DIIAA). The ionic conditions used was 57.5 Na⁺ outside and 12 Na⁺ inside. E) Detail of DIIAA the gating and ionic current components. The red and blue shadowing indicate the presence of gating and ionic currents respectively.

Figure 9:

A general model for fast inactivation from open or closed states. For simplicity, the multiple-step activation is shown as a single transition between the last closed state and the open state in the horizontal direction (rightward) and vertical transitions represent the inactivation path (downward). ➀ represents the Nav channel in the last closed state. ➁ represents the channel with the VSD of DI-DIII in the active (up) position, DIV VSD is in the resting (down) position and a conducting pore: an open channel. Transitions from ➀ to ➂ and ➁ to ➃ occur when DIV VSD moves up, exposing the binding pocket for the IFM motif outside the pore region. Once the binding pocket becomes available, the IFM motif binds and transitions from ➂ to ➄ and ➃ to ➅ occur. The binding of the IFM motif triggers conformational changes that are conducted to the pore region closing the two-layered fast inactivation gate, leading to the closed inactivated state in ➆ or the open inactivated state in ➇.