Conformational dynamics underlying slow inactivation in voltage-gated sodium channels

Area of Science:

• Molecular and Cellular Neuroscience
• Biophysics

Background:

• Voltage-gated sodium (Nav) channels are essential for action potential propagation.
• Slow inactivation, a decrease in Nav channel availability over seconds to minutes, regulates cellular excitability.
• The precise mechanisms of slow inactivation, including selectivity filter and gate coupling, remain incompletely understood.

Purpose of the Study:

• To investigate the conformational dynamics of the Nav channel selectivity filter during slow inactivation.
• To identify molecular determinants coupling the selectivity filter to the primary gate.
• To elucidate the structural basis of Nav channel slow inactivation.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) to monitor selectivity filter conformational changes.
• Electrophysiology and crystallography to assess channel function and structure.
• Site-directed mutagenesis to probe residue function.

Main Results:

• smFRET revealed three distinct conformational states of the NavAb selectivity filter, with high-FRET states enriched by activating voltages, potentially linked to slow inactivation.
• The L176 residue in the P1 helix was identified as a critical coupler between the primary and slow inactivation gates.
• Mutations at L176 and C-terminal deletions altered selectivity filter conformations and modulated slow inactivation kinetics, as did the blocker lidocaine.

Conclusions:

• Slow inactivation in Nav channels is underpinned by selectivity filter pore collapse, visualized as a high-FRET conformation.
• The L176 residue and T206 residue couple conformational changes between the selectivity filter and the primary gate.
• These findings provide molecular insights into the gating mechanisms of Nav channel slow inactivation.