Molecular mechanisms of Slo2 K⁺ channel closure

Abstract

Large conductance K -selective Slo2 channels are in a closed state unless activated by elevated [Na ] . Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss-crossing of the cytoplasmic ends of the S6 α-helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo-electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss-crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively-charged Glu did not induce constitutive channel opening.

Structure-Based Identification and Characterization of Inhibitors of the Epilepsy-Associated KNa1.1 (KCNT1) Potassium Channel

“…Although the cryo-EM structures indicate that activation by sodium involves an expansion of the intracellular pore vestibule , functional experiments with this and the closely related K Na 1.2 and K Ca 1.1 (BK Ca ) channels point to the selectivity filter and proximal hydrophobic residues, rather than an S6 helix bundle, as the location of the channel gate (Garg et al, 2013;Giese et al, 2017;Jia et al, 2018;Suzuki et al, 2016). This means that the inhibitors described here block at the channel gate and this should be a mode of inhibition that is efficacious with virtually all clinical gain-of-function mutations, independent of the mechanism by which increased open probability is achieved, rather than an inhibitor that binds to modulatory sites.…”

Structure-based identification and characterisation of novel inhibitors of KNa1.1 potassium channels, a stratified target for KCNT1-related epilepsy

“…Whilst the cryo-EM structures indicate that activation by sodium involves an expansion of the intracellular pore vestibule (Hite & MacKinnon, 2017), functional experiments with this and the closely-related KNa1.2 and KCa1.1 (BKCa) channels point to the selectivity filter and proximal hydrophobic residues, rather than an S6 helix bundle, as the location of the channel gate (Garg et al, 2013;Suzuki et al, 2016;Giese et al, 2017;Jia et al, 2018). This means that the inhibitors described here block at the channel gate and this should be a mode of inhibition that is efficacious with virtually all clinical gain-of-function mutations, independent of the mechanism by which increased open probability if achieved, rather than an inhibitor that binds to modulatory sites.…”

Targeting KNa1.1 channels in KCNT1-associated epilepsy

“…The exact mechanisms of channel gating have not been elucidated; there is a narrowing of the intracellular pore vestibule by movement of the S6 helices to the 'closed', Na + -unbound state, but there remains sufficient access to the selectivity filter by K + ions [27]. This, and other recent functional studies of K Na 1.1 and the closely related K Na 1.2, point away from canonical S6 helix bundle-crossing as the mechanism of activation gating [48,49]. Instead, it is possible that the channel is gated by a hydrophobic gating mechanism or by a selectivity filter gating mechanism.…”