Conformational dynamics underlying slow inactivation in voltage-gated sodium channels

Voltage-gated sodium (Nav) channels initiate and propagate action potentials in many excitable cells. Upon repetitive activation, the conductance of Nav channels gradually decreases on a timescale ranging from seconds to minutes, a phenomenon known as slow inactivation, which is crucial for regulating the excitability of many cells. Many studies indicated that slow inactivation is associated with conformational changes at the selectivity filter, but the underlying mechanisms remain unclear. By examining the conformational dynamics of a prokaryotic NavAb channel using single-molecule FRET (smFRET), our work revealed the transitions of its selectivity filter among three distinct conformational states and showed that activating voltages enriched the high-FRET conformations, potentially associated with slow inactivation. We further identified L176 in the selectivity filter P1 helix and T206 in the pore-forming S6 helix as residues coupling the primary and slow inactivation gates by showing that the additional L176F mutation stabilizes the S6 C-terminal deletion opening mutant in the closed state. Consistently, our smFRET results also indicated that the high FRET conformation of the selectivity filter was markedly attenuated in the S6 C-terminal deletion opening mutant, but reverted by the L176F mutation. Open-pore blocker lidocaine has been shown to prevent eukaryotic Nav channels from entering the slow inactivation state. Moreover, our smFRET studies showed that it diminished the high FRET conformation of the NavAb selectivity filter in a dose-dependent manner, while the L176F mutation, again, markedly reversed the lidocaine effects. Collectively, our studies suggested that slow inactivation in the NavAb channel results from the collapse of the selectivity filter pore, as revealed by the high FRET conformation in our smFRET measurements. The L176 in the selectivity filter and T206 in the pore-forming S6 helix coordinate conformational changes of the slow inactivation gate at the selectivity filter and the primary gate at the helix bundle crossing, providing the structural basis for slow inactivation in prokaryotic voltage-gated sodium channels.