Mechanisms Underlying C-type Inactivation in Kv Channels: Lessons From Structures of Human Kv1.3 and Fly Shaker-IR Channels

Abstract

Voltage-gated potassium (Kv) channels modulate the function of electrically-excitable and non-excitable cells by using several types of "gates" to regulate ion flow through the channels. An important gating mechanism, C-type inactivation, limits ion flow by transitioning Kv channels into a non-conducting inactivated state. Here, we highlight two recent papers, one on the human Kv1.3 channel and the second on the Drosophila Shaker Kv channel, that combined cryogenic electron microscopy and molecular dynamics simulation to define mechanisms underlying C-type inactivation. In both channels, the transition to the non-conducting inactivated conformation begins with the rupture of an intra-subunit hydrogen bond that fastens the selectivity filter to the pore helix. The freed filter swings outwards and gets tethered to an external residue. As a result, the extracellular end of the selectivity filter dilates and K⁺ permeation through the pore is impaired. Recovery from inactivation may entail a reversal of this process. Such a reversal, at least partially, is induced by the peptide dalazatide. Binding of dalazatide to external residues in Kv1.3 frees the filter to swing inwards. The extracellular end of the selectivity filter narrows allowing K⁺ to move in single file through the pore typical of conventional knock-on conduction. Inter-subunit hydrogen bonds that stabilize the outer pore in the dalazatide-bound structure are equivalent to those in open-conducting conformations of Kv channels. However, the intra-subunit bond that fastens the filter to the pore-helix is absent, suggesting an incomplete reversal of the process. These mechanisms define how Kv channels self-regulate the flow of K⁺ by changing the conformation of the selectivity filter.

Keywords: C-type inactivation; Kv1.3; Shaker-IR; cryo-EM; dalazatide; hydrogen bond network; slow inactivation; voltage-gated potassium (Kv) channels.

FIGURE 1

Structures of Kv channels in open-conducting, C-type open-inactivated and peptide-bound conformations. (A) Structure of KvChim (PDB:2R9R) viewed from the extracellular (left) and membrane planes (middle). The pore domain (PD) shown on the right is formed by S5 and S6 helices together with a P-loop consisting of a turret, pore-helix, SF and loop-to-S6. In the channel’s pore, the inner gate, central cavity, SF, outer vestibule, hinge and S4-S5 linker are shown. (B) Amino acid sequence alignment of P-loops of KvChim, Shaker-IR, Shaker-IRW434F, human Kv1.3 and bacterial KcsA. Residues involved in hydrogen bond networks are highlighted. (C–G) Hydrogen-bond networks in KvChim, PDB:2R9R; Shaker-IR, PDB:7SIP; Shaker-IR W434F, PDB:7SJ1; Apo-Kv1.3, PDB:7WF3, and Dal-Kv1.3, PDB:7WF4. Individual subunits are colored cyan, blue, red, and yellow. Selected residues are shown for clarity. Distances between hydrogen-bonded residues are shown. K⁺ (purple spheres) are shown at K⁺-binding sites, which are numbered.

FIGURE 2

Dimensions of the selectivity filter and a schematic model of C-type inactivation. (A–H) Distances between carbonyl O atoms of residues in SF in KvChim, PDB:2R9R; Shaker-IR, PDB:7SIP; Shaker-IR W434F, PDB:7SJ1; Apo-Kv1.3, PDB:7WF3; Dal-Kv1.3, PDB:7WF4; KcsA open-conducting, PDB:3B5F; I₁ open-inactivated KcsA, PDB:3F7Y; I₂ open-inactivated KcsA, PDB:3F5W. Only two subunits are displayed for clarity. K⁺ (purple spheres) are shown at K⁺-binding sites, which are numbered. (I) Structure-based schematic model of closed, open and C-type inactivated states. The closed model is based on bacterial K⁺ channel KcsA (PDB:1K4C), the open-conducting model on Shaker-IR (PDB:7SIP) and Dal-Kv1.3 (PDB:7WF4), and the C-type open-inactivated model on Apo-Kv1.3 (PDB:7WF4) and Shaker-IR W434F (PDB:7SJ1).