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.
Keywords: Hydrogen sulfide; K+ channel inactivation; Kcna4; Kcnc4; Reactive sulfur species; Sulfhydration.