Modulation of K⁺ channel N-type inactivation by sulfhydration through hydrogen sulfide and polysulfides

Fast N-type inactivation of voltage-gated K⁺ (Kv) channels is important in fine-tuning of cellular excitability. To serve diverse cellular needs, N-type inactivation is regulated by numerous mechanisms. Here, we address how reactive sulfur species-the gaseous messenger H₂S and polysulfides-affect N-type inactivation of the mammalian Kv channels Kv1.4 and Kv3.4. In both channels, the H₂S donor NaHS slowed down inactivation with varying potency depending on the "aging" of NaHS solution. Polysulfides were > 1000 times more effective than NaHS with the potency increasing with the number of sulfur atoms (Na₂S₂ < Na₂S₃ < Na₂S₄). In Kv1.4, C13 in the N-terminal ball domain mediates the slowing of inactivation. In recombinant protein exposed to NaHS or Na₂S₄, a sulfur atom is incorporated at C13 in the protein. In Kv3.4, the N terminus harbors two cysteine residues (C6, C24), and C6 is of primary importance for channel regulation by H₂S and polysulfides, with a minor contribution from C24. To fully eliminate the dependence of N-type inactivation on sulfhydration, both cysteine residues must be removed (C6S:C24S). Sulfhydration of a single cysteine residue in the ball-and-chain domain modulates the speed of inactivation but does not remove it entirely. In both Kv1.4 and Kv3.4, polysulfides affected the N-terminal cysteine residues when assayed in the whole-cell configuration; on-cell recordings confirmed that polysulfides also modulate K⁺ channel inactivation with undisturbed cytosol. These findings have collectively identified reactive sulfur species as potent modulators of N-type inactivation in mammalian Kv channels.