Conformational plasticity of NaK2K and TREK2 potassium channel selectivity filters

Fig. 1. NaK and NaK2K selectivity filter dynamics. A Strategy for introducing two free cysteines within the tetrameric NaK channel. Organization of the tandem dimeric construct where the cysteine is introduced at position T73 at the top part of the first subunit (S1) TM2 domain (PBD 3E8H). Every linker contains two GGGS amino acid repeats. B Single-molecule FRET imaging of NaK proteins in liposomes labeled with Alexa Fluor 555/647 c2 maleimide pair. Proteoliposomes were immobilized on a PEG-coated coverslip surface with biotin-POPE, and then permeabilized by 50 μM β-escin to achieve symmetrical ionic conditions. C Representative donor (blue), acceptor (red) and smFRET (dark blue) traces for NaK T73C and NaK2K T73C, as well as FRET contour plots and histograms (n = 536 and 529 NaK traces; 444 and 303 NaK2K traces, in the presence of 150 mM NaCl or KCl, respectively).

Fig. 2. Permeant ion-dependent kinetics of NaK2K and NaK SF. A Concatenated (blue) and idealized (black) smFRET trajectories in K+ and Na+ for NaK2K. Histograms of experimental and idealized FRET distributions are shown to the right. B Calculated Lifetimes from the idealized traces shows a decrease in high FRET states for NaK2K in Na+. K+ compared with Na+ FRET lifetimes (Low FRET p = 0.6276; Medium FRET p = 0.0640; High FRET p = 0.0096). C Concatenated (gray) and idealized (black) smFRET trajectories in K+ and Na+ for NaK. Histograms of experimental and idealized FRET distributions are shown to the right. D Calculated Lifetimes from the idealized traces do not show any ion dependence of FRET states for NaK. K+ compared with Na+ FRET lifetimes (Low FRET p = 0.1521; Medium FRET p = 0.3376; High FRET p = 0.5376). In (B, D), N = number of sub datasets. Error bars indicate s.e.m. **p < 0.01 vs. Na+. Comparisons between Na+ and K+ analyzed by unpaired t-test. Source data are provided as a Source Data file.

Fig. 3. NaK2K SF conformational changes in cellular membranes. A Chemical structure of ANAP inserted using the amber codon at positions V45 (TM1), A53 (PH) and G76 (TM2) in NaK and NaK2K (PBD 3E8H and 3OUF, respectively, with C-terminal FCYENE sequence motif expressed in CosM6 cells (left) and representative (for at least n = 3 vesicles in each case) images of negative control giant plasma membrane vesicles (GPMVs) where the pANAP vector was not added, and positive GPMVs expressing ANAP-incorporated NaK (right). Pseudocolors for ANAP were used. B Representative emission spectra (red, 150 mM NaCl bath solution; blue, solution containing 150 mM KCl) of ANAP-incorporated NaK and NaK2K mutants. C Summary of shifts in ANAP emission peak at different incorporation sites (for each ANAP-incorporation site, n patches are indicated in each condition) in 150 mM Na+ or K+. Error bars indicate s.e.m. *p < 0.05 vs. Na+ and **p < 0.01 vs. Na+. Comparisons between Na and K analyzed by unpaired t-test. K+ compared with Na+ ANAP emission peaks (NaK2K V45ANAP p = 0.0492; NaK2K A53ANAP p = 0.3589; NaK2K G76ANAP p = 0.0081; NaK V45ANAP p = 0.0558; NaK A53ANAP p = 0.7479; NaK G76ANAP p = 0.1863). Source data…

Fig. 4. Restricting extracellular SF flexibility reduces ion selectivity of NaK2K. A Cartoon representation of the proposed crosslink location between T73C residues (top of TM2) in NaK2K using a 6 nm crosslinker to restrict the extracellular SF flexibility. B NaK2K T73C single-channel recordings in symmetrical 200 mM K+ (blue) and symmetrical 200 mM Na+ (red) without and with the crosslinker (light and dark color, respectively). C NaK2K single-channel K+ conductance is reduced after crosslinking (blue bars), whereas Na+ conductance is increased (red bars). NaK2K open probability in K is reduced after crosslinking (blue bars). Crosslinked samples compared with Control (K+ conductance p = 0.0033; Na+ conductance p = 0.0035; Po in K+ p = 0.0194; Po in Na+ p = 0.3688). D Current-voltage relationships for multi-channel recordings in asymmetrical 200 mM K+ (bath) and 200 mM Na+ (pipette) without and with the 6 nm crosslinker shows significant left shift of reversal potential with the crosslinker, reflecting reduced ion selectivity in NaK2K. Error bars indicate s.e.m. in (C, D). In (C), **p < 0.01 vs. Control. *p < 0.05 vs. Control. ns (not significant), n patches are indicated in each…

Fig. 5. Ion-dependent selectivity filter in TREK2. A Cysteine to Alanine mutations to obtain cysteine-less TREK2 mutant. B Strategy for introducing two free cysteines within the dimeric TREK2 cysteine-less channel. The cysteine is introduced at position S105 at the top part of the first subunit (S1) TM1 domain. Domain organizations of the resulting proteins labeled with Alexa Fluor 555/647 c2 maleimide pair (PBD 4BW5). C Representative smFRET traces for TREK2 S105C, as well as FRET contour plots and histograms (n = 520 and 290 TREK2 traces, in the presence of 150 mM NaCl or KCl, respectively). D Concatenated (mauve) and idealized (black) smFRET trajectories in K+ and Na+ for TREK2. Histograms of experimental and idealized FRET distributions are shown to the right. E Calculated Lifetimes from the idealized traces shows a decrease in high FRET states for TREK2 in Na+. K+ compared with Na+ FRET lifetimes (Low FRET p = 0.5951; Medium FRET p = 0.2164; High FRET p = 0.0038). In blue, D, donor and in red A, acceptor. n = number of molecules. N = number of sub datasets. Error bars indicate s.e.m. **p < 0.01 vs. Na+. Comparisons between Na+ and K+ analyzed by unpaired t-test. Source data are…