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Structural basis for KCNE3 modulation of potassium recycling in epithelia

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Science Advances  09 Sep 2016:
Vol. 2, no. 9, e1501228
DOI: 10.1126/sciadv.1501228
  • Fig. 1 The KCNE3 protein and its function.

    (A) Role of the KCNQ1-KCNE3 channel complex in chloride ion secretion. KCNE3 modulates the voltage-gated potassium channel KCNQ1, removing voltage-dependent gating, leading to a constitutively open leak channel. The KCNQ1-KCNE3 complex is expressed in basolateral epithelial membranes, where it plays a role in K+ recycling necessary for Cl secretion across the apical membrane. Disruptions in transepithelial Cl transport are involved in human pathologies, such as CF and cholera. (B) Sequence and membrane topology of KCNE3. The α-helical regions determined by NMR are highlighted in light green. Sites of disease-linked mutations are highlighted in red, whereas yellow sites were mutated to cysteine and spin-labeled to enable PRE NMR distance measurements. SDSL, site-directed spin labeling; PKCδ, protein kinase Cδ.

  • Fig. 2 Assessment of KCNE3 topology in bicelles from Gd3+ and 16-DSA paramagnet-induced NMR peak broadening.

    1H-15N TROSY NMR (800 MHz) peak intensity reductions induced by the lipophilic 16-DSA and hydrophilic Gd-DTPA paramagnetic probes relative to a matched paramagnet-free reference spectrum are shown as bar graphs in black and salmon, respectively. All three segments with helical secondary structure have some degree of protection from Gd-DTPA and access to 16-DSA. Measurements were carried out for KCNE3 solubilized in DHPC/DMPG bicelles at pH 6.5 and 40°C. TM, transmembrane.

  • Fig. 3 Structure of KCNE3 in a bilayer membrane.

    (A) Representative example from final KCNE3 structure ensemble output from experimentally restrained AMBER molecular dynamics calculations. Only the TMD is shown, as embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer. Residues Thr71, Ser74, and Gly78 are depicted as spheres to highlight the concave face of KCNE3. (B) Same as (A) but with a different view, which includes the surface-associated helices flanking the TMD. (C) Same as (B) but with side chains also shown. The ensemble of 10 final models from the restrained dynamics simulation in a membrane environment is shown in fig. S7.

  • Fig. 4 DEER EPR–based distance measurements for KCNE3 in different model membrane conditions confirm the persistently curved nature of its TMD.

    X-band DEER spectroscopy time evolutions are shown for double spin-labeled KCNE3 at the TMD ends (residues Ser57Cys and Ser82Cys) in different model membranes. (Inset) Distance distributions between the spin labels calculated from the DEER data. The average distances in the different membrane environments are shown with the given uncertainties reflecting the SEs from fitting of the data with a 95% confidence interval. DEER EPR data show similar TMD distance measurements taken in LMPC micelles, DHPC/DMPG bicelles, or POPC/POPG bilayers.

  • Fig. 5 The S4 domain of KCNQ4 causes KCNQ1 to be inhibited by KCNE3.

    Average whole-cell currents recorded from cells transiently expressing KCNQ1/KCNQ4 chimeras alone (+DsRed) or with KCNE3: (A) KCNQ1 + KCNE3, (B) KCNQ4 + KCNE3, (C) KCNQ1 with the S1 domain of KCNQ4, (D) KCNQ1 with the S2 domain of KCNQ4, (E) KCNQ1 with the S3 domain of KCNQ4, (F) KCNQ1 with the S4 domain of KCNQ4, (G) KCNQ1 with the S5 domain of KCNQ4, and (H) KCNQ1 with the S6 domain of KCNQ4.

  • Fig. 6 Pairwise replacement of sites 240 + 244 or 241 + 244 in the S4 domain of KCNQ1 with the corresponding residues of KCNQ4 causes KCNQ1 to be inhibited by KCNE3.

    Average whole-cell currents recorded from cells transiently expressing KCNQ1 mutants or KCNQ4 chimeras, both alone (+DsRed) and with KCNE3: (A) KCNQ1 with the S4 domain of KCNQ4 (KCNQ1[Q4S4]); (B) Leu239Val,His240Arg,Val241Met,Gln244Arg KCNQ1; (C) Leu239Val,His240Arg,Val241Met KCNQ1; (D) His240Arg,Gln244Arg KCNQ1; (E) Val241Met,Gln244Arg KCNQ1; and (F) Gln244Arg KCNQ1. We note that the His240Arg, Gln244Arg KCNQ1, and Gln244Arg KCNQ1 mutants have been previously examined (36), with somewhat different results being obtained. However, our studies were conducted using CHO cells, which are bereft of endogenous expression of KCNE subunits, whereas the previous studies were conducted using KCNQ1 expressed in oocytes, in which the expression of either endogenous KCNE subunits or endogenous K+ channels can confound studies of potassium channels (7577).

  • Fig. 7 Oxidation state–dependent electrophysiology measurements confirm that KCNQ1 Leu142 and KCNE3 Met59 residues are in close contact, as are KCNE3 Ser82 and KCNQ1 Gln244.

    (A) Average whole-cell currents recorded from cells transiently expressing KCNQ1 Leu142Cys plus KCNE3 Met59Cys exposed to control bath solution, +DTT, or Cu-phenanthroline (Cu-phen.). The solid line between the two traces indicates the zero current. (B) Voltage dependence of activation for currents recorded from cells expressing KCNQ1 Leu142Cys plus KCNE3 Met59Cys exposed to control bath solution (○), +DTT (▪), or Cu-phenanthroline (♦). (C) Average current-voltage (I-V) relationships normalized by membrane capacitance measured from cells expressing KCNQ1 Leu142Cys plus KCNE3 Met59Cys exposed to control bath solution (○), +DTT (▪), or Cu-phenanthroline (♦). (D) The KCNQ1 Cys122Ser/Gln244Cys + KCNE3 Ser82Cys complex is sensitive to DTT, suggesting that KCNQ1 Gln244Cys and KCNE3 Ser82Cys are in close enough contact with each other to spontaneously form a disulfide bond. (E) Average I-V normalized by membrane capacitance measured from cells expressing WT KCNQ1 plus KCNE3 Ser82Cys exposed to control bath solution (○), +DTT (▪), or Cu-phenanthroline (♦). (F) Average I-V normalized by membrane capacitance measured from cells expressing KCNQ1 Cys122Ser/Gln244Cys plus KCNE3 Ser82Cys exposed to control bath solution (○), +DTT (▪), or Cu-phenanthroline (♦). I tailmax, tail current maximum.

  • Fig. 8 Activated-state KCNE3-KCNQ1 channel complex derived from experimentally restrained docking.

    The lowest-scoring complex is displayed with KCNQ1 colored blue at residues 120 to 244 (voltage-sensor domain), green at residues 245 to 259 (S4-S5 linker), and red at residues 260 to 370 (pore); KCNE3 is colored gold. The top image represents the view looking from the extracellular space toward the membrane plane. The middle image is the view from within the membrane plane and is zoomed in on the site of KCNE3-KCNQ1 interaction. The bottom image is viewed from slightly above the membrane plane, with the extracellular environment above and the intracellular environment below.

  • Fig. 9 Reversal of estrogen-induced inhibition of KCNQ1-KCNE3 association by Q244R KCNQ1 mutation and structural model to explain this effect.

    (A) (Left) Average whole-cell currents recorded from cells transiently expressing KCNQ1 WT plus KCNE3 exposed to control solution (ethanol) or estrogen (17β-estradiol, 100 μM). The solid line between the two traces is at zero current. (Right) Average I-V normalized by membrane capacitance measured from cells coexpressing KCNQ1 WT plus KCNE3 WT in the absence (▪, n = 16) or presence of estrogen (▴, n = 14). ***P ≤ 0.001, *P ≤ 0.05. (B) (Left) Average whole-cell currents recorded from cells transiently coexpressing KCNQ1 Gln244Arg and KCNE3 WT exposed to control solution or to estrogen (right). (Right) Average I-V normalized by membrane capacitance measured from cells coexpressing KCNQ1 Gln244Arg with KCNE3 WT in the absence (▪, n = 13) or presence of estrogen (▴, n = 13). Note that Fig. 6F provides key control data showing that the Q244R mutation in KCNQ1 induces a marked loss of current when KCNE3 is not present. (C) Cartoon representation of the channel, less one voltage sensor, set on the background of the complete channel shown in translucent spheres. KCNE3 WT, shown as a yellow ribbon, forms an energetically favorable complex with KCNQ1 WT (top). Phosphorylated KCNE3 Ser82 reduces the affinity to the WT channel (middle). When the KCNQ1 Gln244Arg mutation is introduced, the binding affinity to phosphorylated KCNE3 Ser82 is restored (bottom).

  • Fig. 10 Location of key KCNQ1 functional determinants of KCNE3 modulation.

    The integrative models of the KCNE3-KCNQ1 complex are useful to visualize the S4 functional determinants of KCNE3 modulation in KCNQ1. The upper left panel highlights His240, Val241, and Gln244 in cyan that are key to KCNE3 modulation. The upper right panel shows the same residues, zoomed in, with both a cartoon and transparent surface representation of the same region of KCNQ1. A surface representation (lower left) of these KCNQ1 residues (cyan) shows that these residues are surface-accessible in the KCNE3 binding cleft. The lower right panel shows the predicted hydrogen bond between KCNE3 Arg83 and KCNQ1 Gln244, which suggest a potential mechanism for the distinct KCNE3 modulation of KCNQ1 and KCNQ4.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1501228/DC1

    Supplementary Materials and Methods

    fig. S1. Integrative structural biology to generate experimentally restrained structural models of the KCNE3-KCNQ1 channel complex.

    fig. S2. Chemical shift index analysis for KCNE3 in bicelles.

    fig. S3. RDC NMR data for KCNE3 in bicelles.

    fig. S4. Dipolar wave analysis of bicellar KCNE3 1H-15N RDCs.

    fig. S5. Examples of PRE NMR data for KCNE3 in bicelles.

    fig. S6. 15N NMR relaxation measurements for KCNE3 in bicelles.

    fig. S7. Representative structures of KCNE3 amphipathic and transmembrane helices from AMBER restrained molecular dynamics (rMD) simulations.

    fig. S8. Water access to the TMD of KCNE3.

    fig. S9. Sequence conservation between KCNQ1 and KCNQ4.

    fig. S10. Homology modeling of the open state of KCNQ1.

    fig. S11. Homology/Rosetta modeling of the KCNQ1 channel open state.

    fig. S12. Flowchart displaying the process used to dock KCNE3 to open-state KCNQ1.

    fig. S13. Calculated binding energies (ddG) versus interface root mean squared deviation (Irms) of interface α-carbon positions compared to the lowest-scoring KCNE3-KCNQ1 (open) complex.

    fig. S14. Same plot as fig. S13 with color displaying the total distance restraint violations.

    fig. S15. Rebuilding flexible regions within the KCNQ1-KCNE3 complex.

    fig. S16. Representative 23 KCNE3-KCNQ1 models based on satisfaction of experimental restraints and Rosetta scoring function.

    fig. S17. Comparison between structurally characterized KCNE family members.

    fig. S18. Sequence conservation within the entire KCNE family and within KCNE3 from different organisms.

    fig. S19. KCNE3 reduces the KCNQ1[Q4S4] current amplitude without reducing channel protein levels at the membrane.

    table S1. Statistics for restraints, structural calculations, and structural quality for the 10 lowest-energy structures of 9764 calculated using XPLOR and further refined in AMBER.

    table S2. KCNQ1-KCNE3 residue pairs predicted to be proximal based on experimental work.

    PDB coordinates

    References (7883)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Integrative structural biology to generate experimentally restrained structural models of the KCNE3-KCNQ1 channel complex.
    • fig. S2. Chemical shift index analysis for KCNE3 in bicelles.
    • fig. S3. RDC NMR data for KCNE3 in bicelles.
    • fig. S4. Dipolar wave analysis of bicellar KCNE3 1H-15N RDCs.
    • fig. S5. Examples of PRE NMR data for KCNE3 in bicelles.
    • fig. S6. 15N NMR relaxation measurements for KCNE3 in bicelles.
    • fig. S7. Representative structures of KCNE3 amphipathic and transmembrane helices from AMBER restrained molecular dynamics (rMD) simulations.
    • fig. S8. Water access to the TMD of KCNE3.
    • fig. S9. Sequence conservation between KCNQ1 and KCNQ4.
    • fig. S10. Homology modeling of the open state of KCNQ1.
    • fig. S11. Homology/Rosetta modeling of the KCNQ1 channel open state.
    • fig. S12. Flowchart displaying the process used to dock KCNE3 to open-state KCNQ1.
    • fig. S13. Calculated binding energies (ddG) versus interface root mean squared deviation (Irms) of interface α-carbon positions compared to the lowest-scoring KCNE3-KCNQ1 (open) complex.
    • fig. S14. Same plot as fig. S13 with color displaying the total distance restraint violations.
    • fig. S15. Rebuilding flexible regions within the KCNQ1-KCNE3 complex.
    • fig. S16. Representative 23 KCNE3-KCNQ1 models based on satisfaction of experimental restraints and Rosetta scoring function.
    • fig. S17. Comparison between structurally characterized KCNE family members.
    • fig. S18. Sequence conservation within the entire KCNE family and within KCNE3 from different organisms.
    • fig. S19. KCNE3 reduces the KCNQ1Q4S4 current amplitude without reducing channel protein levels at the membrane.
    • table S1. Statistics for restraints, structural calculations, and structural quality for the 10 lowest-energy structures of 9764 calculated using XPLOR and further refined in AMBER.
    • table S2. KCNQ1-KCNE3 residue pairs predicted to be proximal based on experimental work.
    • References (7883)

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