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Structural dynamics of potassium-channel gating revealed by single-molecule FRET

Nature Structural & Molecular Biology volume 23, pages 31–36 (2016)Cite this article

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Abstract

Crystallography has provided invaluable insights regarding ion-channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites and then examined structural dynamics within lipid membranes by single-molecule fluorescence resonance energy transfer (FRET). We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states, both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and in cation channels in general.

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Figure 1: PIP2-dependent gating of KirBac1.1-channel activity.
Figure 2: smFRET measurements of the KirBac1.1 channel in liposomes.
Figure 3: Conformational fluctuations of KirBac1.1 in liposomes.
Figure 4: Motion of the TM with respect to CTD upon PIP2 gating.
Figure 5: Conformational dynamics of KirBac1.1- and PIP2-dependent gating.

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Acknowledgements

Financial support was provided by US National Institutes of Health (NIH) grant HL54171 (C.G.N.) and US National Science Foundation grant PHY1430124 (T.H.). T.H. is supported as an investigator of the Howard Hughes Medical Institute. W.F.B. was supported by NIH grants T32 HL007275 and T32 HL125241.

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Author notes

  1. Shizhen Wang and Reza Vafabakhsh: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology and Physiology, Center for Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, Missouri, USA

    Shizhen Wang, William F Borschel & Colin G Nichols

  2. Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Reza Vafabakhsh & Taekjip Ha

  3. Howard Hughes Medical Institute, Baltimore, Maryland, USA

    Taekjip Ha

  4. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland, USA

    Taekjip Ha

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  1. Shizhen Wang
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Contributions

S.W., R.V., T.H. and C.G.N. conceived and designed the experiments; S.W. and W.F.B. performed the electrophysiological studies; S.W. designed, constructed, purified and labeled the protein samples with fluorophore; W.F.B. analyzed the single-channel recordings; and S.W. and R.V. collected and analyzed the smFRET data. The paper was written by S.W. and C.G.N. and edited by R.V., W.F.B. and T.H.

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Correspondence to Taekjip Ha or Colin G Nichols.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Purified KirBac1.1 mutants for smFRET experiments.

(A) Gel filtration profiles of KirBac1.1 mutants labeled with Alexa Fluor 555 and Alexa Fluor 647 c2 maleimide. Purified KirBac1.1 proteins (1 mg/ml) were reacted with 1:1 mixer of Alexa Fluor 555 and Alexa Fluor 647 c2 maleimide at protein/fluorophore ratio of 1:4 for 2 hrs under room temperature. The labeled mixture was loaded onto a metal affinity column and the non-covalently attached fluorophores were removed by extensive washes. The labeled proteins were eluted using imidazole and then loaded onto a gel filtration column. The central fractions (between the dash lines) of tetramer peaks were collected for functional study (rubidium flux assay) or smFRET experiments. (B) Channel function assay of KirBac1.1 mutants labeling with Alexa Fluoro 555 and Alexa Fluoro 647 by rubidium flux. Mutant proteins were labeled with 1:1 mixer of Alexa Fluoro 555 and Alexa Fluoro 647 and reconstituted into liposomes at a protein:lipid ratio of 1:100. The lipids used for reconstitution were POPE and POPG (3:1), with or without 1% PIP2. The intraliposomal solution contained 20 mM HEPES, 450 mM KCl, 1 mM EDTA, 1 mM EGTA, 4 mM NMDG, pH7.5. The extraliposomal solution contained 20 mM HEPES, 400 mM Sorbitol, 1 mM EDTA, 1 mM EGTA, 4 mM NMDG, pH7.5. The rubidium assay was performed following the protocol described previously (19), and all rubidium uptakes were normalized against WT. The data are presented as mean±se, n=3. The labeling efficiencies of protein samples were determined by a Nanodrop 1000 spectrophotometer according to the protocol provided with the Alexa Fluor 555 and 647 dye package. The labeling efficiencies were location-dependent and changed slightly for different batches. For construct A45C-WT and T120C-WT constructs, the typical labeling efficiencies were 0.6~0.7; for W48C-WT construct, the labeling efficiency was ~0.5; for A167C-WT and A273C-WT, the labeling efficiencies were typically >0.9. (C) The specificity of fluorophore labeling in KirBac1.1 cysteine mutants examined by SDS-PAGE. The labeled protein samples were separated by 4-20% SDS-PAGE, and fluorescence images were acquired with a Kodak fluorescence scanner. For visualizing Alexa Fluor 555, the excitation light was 535 nm and emission images were collected with a 600nm long-pass filter; for Alexa Fluor 647, the excitation light was 637 nm, and emission images were collected with a 670 nm long-pass filter. The protein was visualized by Coomassie Brilliant Blue R-250 staining and the images were acquired with a document scanner.

Supplementary Figure 2 Representative smFRET traces.

Representative smFRET traces of T120C-WT (A), A45C-WT (B) and A273C-WT (C) sample without or with PIP2.

Supplementary Figure 3 smFRET histograms and contour plots of KirBac1.1 mutants at 30-ms and 100-ms time resolution.

(A) FRET histograms and contour plots of T120C-WT (n=53 and 40 for control and PIP2 conditions, respectively) and A45C-WT (n=218 and 235 for control and PIP2 conditions, respectively) mutants with single molecule imaging data of 30 ms time resolution. (B) FRET histograms and contour plots of W48C-WT (n=100 and 46 for control and PIP2 conditions, respectively) and F167C-WT mutants (n=333 and 254 for control and PIP2 conditions, respectively).

Supplementary Figure 4 Ensemble FRET measurements of KirBac1.1 mutants labeled at different structural motifs in liposomes with or without PIP2.

KirBac1.1 mutants were labeled with EDANS/DABCYL-plus (A) or AlexaFluor-488/QSY7 FRET pair (B) and reconstituted into liposomes (POPE:POPG = 3:1) with or without 1% PIP2. Ensemble FRET measurements were performed following the protocols as described previously (Wang, S. et al., 2012, Nature communications 3, 617). The data were presented as mean± se, n = 6 (*p<0.05, **p<0.01).

Supplementary Figure 5 Representative smFRET traces.

Representative smFRET traces of T120C/A270C-WT-WT-WT (A), T120C-A270C-WT-WT (B) and T120C-WT-A270C -WT (C) samples without or with PIP2.

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Wang, S., Vafabakhsh, R., Borschel, W. et al. Structural dynamics of potassium-channel gating revealed by single-molecule FRET. Nat Struct Mol Biol 23, 31–36 (2016). https://doi.org/10.1038/nsmb.3138

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