Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain

Abstract

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-EM structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic T1 domain which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the VSD/PD interface. Supported by MD simulations and electrophysiological and mutational analyses, we identify close communication between α6 helix of T1 domain, S4/S5 linker and S6T helix as responsible for the ultra-fast activation/deactivation and open state stabilisation that are unique to Kv3 channels. These findings provide fundamentally new insights into gating control and disease mechanisms and guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

Mechanism of 4-aminopyridine inhibition of the lysosomal channel TMEM175

“…Cryo-EM maps and atomic coordinates for 4-AP bound TMEM175 have been deposited with the European Molecular Data Bank (EMDB, https://www.ebi.ac.uk/emdb/ ) and Protein Data Bank (PDB, https://www.rcsb.org/ ) (accession nos. EMDB-27436 ( 42 ) and PDB 8DHM ( 43 )) Datasets from the PDB used in this study include 2A79 ( 44 ), 6WC9 ( 45 ), 7PHL ( 46 ), 7SSV ( 47 ), and 7UNL ( 48 ). Force field parameters for 4-AP compatible with CHARMM are publicly available at https://github.com/Faraldo-Gomez-Lab-at-NIH/Download .…”

A KCNC1‐related neurological disorder due to gain of Kv3.1 function

“…These three residues are located between the selectivity filter and the conserved PXP motif in S6 (PVP in Kv3 subunits); the PXP motif which forms a “kink” that has been shown to be important for gating of K+ channels 13 and the identified variants could decrease the stability of the closed state and favor channel opening. Recent publication of the cryo‐EM structure of human Kv3.1 provides unprecedented insight into the mechanism of gating of Kv3 channels 14 . The Met430 residue has been shown to contribute to ion conduction and regulate Kv3.1 pore stability as mutation of this residue to a lysine induces channel flicker, 15 while the cryo‐EM structure reveals that the Met430 residue in S6 forms a intersubunit sulfo‐aromatic interaction with the F345 residue in S5 of adjacent subunits.…”

“…Recent publication of the cryo-EM structure of human Kv3.1 provides unprecedented insight into the mechanism of gating of Kv3 channels. 14 The Met430 residue has been shown to contribute to ion conduction and regulate Kv3.1 pore stability as mutation of this residue to a lysine induces channel flicker, 15 while the cryo-EM structure reveals that the Met430 residue in S6 forms a intersubunit sulfo-aromatic interaction with the F345 residue in S5 of adjacent subunits. Why the variant KCNC1-p.Ala421Val located slightly more proximaland which also converts between two small amino acids with hydrophobic side chainsinstead produces a profound LoF (and the more severe DEE) remains unclear.…”

Pentameric assembly of the Kv2.1 tetramerization domain

“…Intriguingly, a recent structural study discovered that the orientation of the T1 domain in Kv3 channels differs from that in structures of the Kv1.2-2.1 paddle chimera. The most C-terminal -helix of the Kv3.1a T1 domain is rotated towards and contacts the linker between the voltage-sensor and pore domains, which may stabilize the pore and contribute to the unique kinetic properties of Kv3 channels (Chi et al, 2021). It remains to be determined whether a similar arrangement occurs in Kv2 channels.…”