Conformational plasticity of NaK2K and TREK2 potassium channel selectivity filters

Abstract

The K+ channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K+ binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K+-selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K+ channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K+ conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K+ channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K+ channels. Our results establish the generality of K+-induced SF conformational stability across the K+ channel superfamily, and introduce an approach to study manipulation of channel selectivity.

From head to tail - Atomistic mechanism of long-range coupling from the cytosolic sensor domain to the selectivity filter in TREK K2Pchannels

“…Particularly for the TREK-2*, a strong hydrogen bonding between the side chains of Y175 (SF1) and T278 (P2) was observed in the up-state simulations, but this interaction was partially diminished in the down-state one. This result is in line with several recent solid-state NMR studies that emphasize the vital roles of the interactions between the SF and the pore helix in stabilizing the conductive SF conformation of K + channels 50,51 . Moreover, the interaction between the P2 and M4 helix is primarily mediated by the interaction between F316 (M4) and T280 (P2), an interaction that was predominant only in the up-state simulations.…”

Cryo-EM structures of Kv1.2 potassium channels, conducting and non-conducting

“…The X-ray crystal structure of the KcsA channel in low K + solution (Zhou et al, 2001) shows a collapsed selectivity filter, in which the residues V26’ and G27’ are rearranged, abolishing the binding sites IS2 and IS3. In low K + other channels including KirBac2.2 and K2p10.1 show large conformational fluctuations of the outer pore in low K+ (Matamoros et al, 2023; Wang et al, 2019), and in the model potassium channel NaK2K the outer half of the selectivity filter unravels at low K + (Sauer et al, 2011). On the other hand, Shaker channels are seen to conduct Na + in the absence of K + (Melishchuk et al, 1998).…”

“…Sauer et al (2011) conclude that the standard four-ion-site, K + selective configuration of potassium channel selectivity filters is an energetically unfavorable, strained backbone conformation, and the weakening of the specific interactions that hold it in place lead to its unraveling, producing a lower-energy configuration that yields a nonselective conduction path. Similarly, the removal of K + ions from NaK2K destabilizes the selectivity filter, resulting in large fluctuations of the outer pore (Matamoros et al, 2023).…”

“…In KcsA the replacement of most K + with Na + results in a collapse of the selectivity filter region, occluding the two central ion-binding sites (Zhou et al, 2001). Other channels including NaK2P and members of the K2P family show a destabilization of the external part of the pore at low K + concentrations, abolishing the two outer ion- binding sites (Lolicato et al, 2020; Matamoros et al, 2023; Sauer et al, 2011). In low potassium the NaK2P channel actually shows a dilation of the outer selectivity filter very similar to that seen in Shaker-W434F and the corresponding K1.2-2.1 mutant.…”

Selectivity filter mutations shift ion permeation mechanism in potassium channels

“…Indeed, we previously observed dilated and more dynamic SFs of non-selective channels that allow simultaneous permeation of ions and water molecules, and we linked this increased water co-permeation to reduced K + /Na + selectivity (10). Of particular interest, a series of FRET experiments on KirBac1.1 (35), NaK2K and TREK-2 (36) channels showed similar, ion-dependent SF conformational states in all of these channels. In these experiments, the presence of K + in the SF promotes a compact, Rb + -permeable conformation of the SF, whereas in Na + the SF adopts a dilated, Na + and water permeable state, thus offering an explanation how water-free direct knock-on can occur via compact states, and yet still display water permeation through Na + -induced dilated conformations.…”