Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

Abstract

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Figure 1. Establishment of Nav1.4-WSW as a suitable background for MTSET accessibility studies.

Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 800 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW channels. For the closed-state modification protocol, channels were held at −130 mV, given a 10-ms test pulse to −30 mV, and then subjected to two seconds of MTSET treatment at −130 mV (orange bar indicates the duration of MTSET perfusion). This process was repeated every 10 s with MTSET washout occurring at −130 mV. Test pulse current traces are plotted before and after each subsequent exposure to MTSET (a, bottom). For the open-state modification protocol, channels were held at −130 mV, given a 10-ms test pulse to −30 mV, and then exposed to MTSET for 50 ms at 0 mV. This process was repeated every 40 s with MTSET washout occurring at −130 mV. Test pulse current traces are plotted before and after each subsequent exposure to MTSET (b, bottom). The brown line was included to highlight any changes in current amplitude. Peak sodium current does not decrease after exposure to MTSET while channels are closed or open, indicating that this mutant does not react with MTSET.

Figure 2. State-dependent modification of L1580C.

Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 200 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW-L1580C channels. Test pulse current traces for closed-state modification (a, bottom) and open-state modification (b, bottom) are plotted before and after each subsequent exposure to MTSET. Peak sodium current does not change when MTSET is applied to closed channels; however, sodium current decreases after each 100 ms exposure of MTSET to open channels.

Figure 3. Changes in accessibility follow channel gating.

The rates of modification (red boxes) in the presence of MTSET perfused on inside-out patches held at different voltages during MTSET exposure are plotted alongside the conductance–voltage relation (black diamonds) for Nav1.4-WSW-L1578C channels. The red vertical error bars represent the s.d. of the modification rates (n=3); the solid black line is a Boltzmann function fit to the mean values for independent fits to three experiments. These data demonstrate that voltage-dependent changes in channel open probability and the rates of modification at different voltages are correlated.

Figure 4. Modification of F1594C.

Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 100 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW-F1594C channels. Test pulse current traces for closed-state modification (a, bottom) and open-state modification (b, bottom) are plotted before and after each subsequent exposure to MTSET. Peak sodium current decreases after each 100 ms exposure of MTSET to both closed and open channels.

Figure 5. State-dependent accessibility of DIV S6 cysteine mutants.

Cysteine modification rates in the closed (filled circles) and open (open circles) states are plotted on a logarithmic scale for positions 1578-1594. Each point is the mean of three experiments. The length of the bar represents the fold change in rate between the closed and open states. Sites above I1590 have closed-state reactivity values <1 M⁻¹ s⁻¹. I1581C, V1582C, N1584C and N1593C displayed no change in peak current after MTSET exposure (red marks). Depending on the rates of modification, the concentration of MTSET used to test each site ranged from 100 μM to 2 mM.

Figure 6. Alignment of pore domains and homology models.

(a) Aligned sequences of individual pore domains for rat Kv1.2, Shaker Kv, rat Nav1.4, rabbit Cav2.1 and the bacterial sodium channels NavAb, NavMs, NavRh and NaChBac (see Results). Red and blue highlighted residues in Nav1.4 DIV, Shaker Kv and Cav2.1 DI-III denote positions accessible to internal MTSET in both closed and open states (blue) or open states only (red). Residues highlighted in grey indicate positions where internal MTSET did not produce a functional change in the peak current response. Shaker Kv and Cav2.1 accessibility data are from previous experiments. Residues aligned with those contributing to the selectivity filter and intracellular pore gate in Nav1.4 are highlighted in yellow. Highly conserved residues are shown on a black background. Grey scale bar below the sequences shows the CORE index reliability score at each position (light grey=low reliability, black=high reliability) along with an indicator for highly conserved (:) and semi-conserved (.) positions. (b) Homology models of the pore lining S6 segments from each domain in rat Nav1.4 channels based on prokaryotic structures in either the closed pore (NavAb, white) or open pore (NavMs, red) conformations. View is from the cytoplasm looking up through the channel pore. The residues comprising the pore gate are shown in stick representation in both their closed (white) and open (yellow) configurations. Images were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger, LLC). (c) The solvent accessible surface at the gate region is shown in red for both the closed pore (left) and open pore (right) Nav1.4 models. View is the same as in b. (d) View of the gate region from within the plane of the membrane for closed (left) and open (right) Nav1.4 models. For clarity, only domains II and IV are shown. The residues contributing to the pore gate in the closed pore model are shown in stick representation and the solvent accessible surface is shown in red. For the open pore model, the solvent accessible surface is transparent so that the gate residues can be visualized.