Conformational dynamics underlying slow inactivation in voltage-gated sodium channels

Abstract

Voltage-gated sodium (Nav) channels initiate and propagate action potentials in many excitable cells. Upon repetitive activation, the fraction of Nav channels available for excitation gradually decreases on a timescale ranging from seconds to minutes, a phenomenon known as slow inactivation. This process is crucial for regulating cellular excitability and firing patterns. Slow inactivation is proposed to result from the collapse of the selectivity filter pore coupled with the opening of the primary helix bundle crossing gate. However, the conformational changes underlying slow inactivation and the molecular coupling between the selectivity filter and primary gate remain unclear. In this study, we investigated the conformational dynamics of the selectivity filter in prokaryotic NavAb channels reconstituted into liposomes using single-molecule FRET (smFRET). Our smFRET data revealed the conformational transitions in the NavAb selectivity filter pore among three distinct states, with activating voltages enriching the high-FRET conformations, potentially associated with slow inactivation. Using electrophysiological and crystallographic methods, we further identified the L176 residue in the selectivity filter P1 helix as a critical coupler between the primary and slow inactivation gates. We showed that L176 mutations with side chains of larger sizes significantly facilitated the slow inactivation of the NavAb channel, and the L176F mutation forced the opening mutant carrying the C-terminal deletion to be crystallized at the closed state. Consistently, our smFRET results revealed that C-terminal deletion markedly attenuated the high FRET conformation of the selectivity filter, which was restored by the L176F mutation. Moreover, using the classical Nav open-pore blocker lidocaine, we showed that it also depleted the high FRET conformation of the NavAb selectivity filter in a dose-dependent manner. The L176F mutation, again, markedly reversed the conformational shifts caused by lidocaine, an effect similar to it on the opening mutant carrying the C-terminal deletion. Our studies consistently suggested that slow inactivation in the NavAb channel is underlined by the collapse of the selectivity filter pore, represented by the high FRET conformation uncovered by our smFRET measurements, while the L176 residue at the P1 helix of the selectivity filter and T206 at the pore lining helix couple the conformational changes of the slow inactivation gate at selectivity filter and the primary gate at the helix bundle crossing.

Extended Figure 1.. The channel activity of NavAb tandem dimers

Representative trace of NavAb (a) BG dimer carrying only the N49K background mutation, (b) BG dimer with additional V190C in first subunit, (c) BG dimer with additional L176F mutation in both subunits, (d) BG dimer with additional L176F mutation in both subunits and V190C in first subunit, and (e) BG dimer with C-terminal deletion ΔC230 in both subunits. All currents were generated by stimulation pulses of −20 mV.

Extended Figure 2.. Activating voltage induces the constricted conformation at the NavAb selectivity filter at the potential inactivation state

a. Representative smFRET traces exhibiting conformational transitions among 3 different states; b-g. Contour maps (b, d and f) and FRET state occupancies (c, e and g) of smFRET data collected from either E189C (b, c, d and e) or V190C (f and g) labeling sites carrying either L176F (d, e, f and g) or T206A (b and c) background mutations, under −85, 0 and 120 mV.

Extended Figure 3.. Voltage-dependent activation and inactivation of BG, L176F, L176W, ΔC230 and ΔC230/L176F mutants.

a-d. Representative traces of deactivation tail currents and steady-state inactivation, and voltage-dependency of activation and inactivation of BG, L176F, L176W and ΔC230 mutants. e. Representative trace of the ΔC230/L176F current generated by stimulation pulses of −20 mV.

Figure 1.. Activating voltage induces the constricted conformation at the NavAb selectivity filter at the potential inactivation state

a. Cartoon of the NavAb pore domain, with 2 subunits removed for clarity (PDB: 5VB2). The pore-forming helices are colored cyan, and the P1/P2 helices of the selectivity filter are colored pink. The C-terminal helices are colored gray. The Cα carbon atoms of E189C and V190C at diagonal subunits for fluorophore labeling were highlighted by orange and blue spheres, respectively. b. The experimental setup to examine conformational dynamics of the NavAb selectivity filter using smFRET. The fluorophore-labeled NavAb channels were reconstituted into liposomes (POPE/POPG=31, w/w), and the proteoliposomes were immobilized on the PEGylated surface through neutravidin and biotinylated anti-histag or anti-FLAG tag antibodies, so only those with the C-terminus of the NavAb channel facing outside were retained for smFRET imaging. Transliposomal electrical potentials were generated by transliposomal K⁺ gradients and valinomycin, with the intraliposomal K⁺ concentration as 5 mM and extraliposomal K⁺ concentration as 150, 5, and 0.04 mM for −85, 0, and 120 mV, respectively. c. smFRET traces showed that FRET between Cy3/Cy5 fluorophores conjugated at E189C or V190C at the P2 helix in the NavAb selectivity filter exhibits transitions among 3 distinctive states. d-e. Contour maps of smFRET traces collected from E189C (d) and V190C (e) labeling sites indicated that strong activating voltages enriched the high FRET populations. f. The kinetic model for analyzing smFRET traces collected from E189C and V190C labeling sites. smFRET traces were idealized into low, medium and high FRET states with peak centers at 0.25, 0.55 and 0.8. The FRET state occupancy data clearly showed that strong activating voltages enriched the high FRET state but diminished the medium and low FRET states.

Figure 2.. Mutations at the selectivity filter or C-terminus change the inactivation of the NavAb channel

a. Steady-state inactivation of NavAb BG, L176F, L176W, and ΔC230 mutants. b. The electron density map of BG, L176F, ΔC230 and ΔC230/L176F mutants around L176 in the selectivity filter and T206 in the S6 helix. The 2F_O − F_C electron density map contoured at 1σ (blue mesh) shows the proximity between the S6 helix and the selectivity filter. c. Root Mean Square Deviation (RMSD) of pore domain residues of mutant channels against NavAb BG mutant carrying only the N49K background mutation. d. Superimposition of the selectivity filter of crystal structures of the BG, L176F, L176W, ΔC230 and ΔC230/L176F mutants.

Figure 3.. The selectivity filter structure and communication between the S6 helix and the selectivity filter

a. Superimposition of the pore domains of NavAb BG, L176F, ΔC230 and ΔC230/L176F mutants. b. Pore diameter profiles of NavAb BG, L176F, ΔC230 and ΔC230/L176F mutants. c. Superimposition of the selectivity filter and the S6 helix of BG and ΔC230 mutants. d. Superimposition of the selectivity filter and the S6 helix of BG, L176F and ΔC230/L176F mutants. e. Superimposition of the selectivity filter and the S6 helix of L176F and L176W mutants.

Figure 4.. Crosstalk between the C-terminal primary gate and the selectivity filter

a-d. Contour maps (a, c) and FRET state occupancies (b, d) of smFRET data collected from V190C labeling sites carrying the ΔC230 C-terminal deletion, without (a, b) or with (c, d) the L176F mutation, under −85, 0, and 120 mV. Contour maps of smFRET data collected from V190C labeling sites carrying the ΔC230 C-terminal deletion and L176F mutation. e-f. Contour maps (e) and FRET state occupancies (f) of the smFRET data collected from the V190C labeling sites without (BG) or with the L176F mutation in the presence of different concentrations of lidocaine under 0 mV. Lidocaine was shown to deplete the high FRET population in a dose-dependent manner, while the L176F mutation, which enhances slow inactivation, restored the high FRET conformation of the selectivity filter.

Figure 5.. Conformational transitions in the NavAb selectivity filter from conductive state to early and late inactivated states revealed by smFRET measurements

The low, medium, and high FRET populations correspond to the dilated/conductive, intermediate/early and constricted/late slow inactivation states, respectively. T206A, lidocaine, and C-terminal deletion enriched conductive state conformations, while L176F, C-terminal helix bundle and activating voltages promoted early and late slow inactivated state conformations. Only the pore domains of two subunits were shown, with the front and rear subunits removed for clarity.