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Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations

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Science Advances  07 Mar 2018:
Vol. 4, no. 3, eaar2631
DOI: 10.1126/sciadv.aar2631
  • Fig. 1 KCNQ1 expression levels and surface trafficking efficiencies.

    Data are color-coded: LQTS mutant (red), VUS (blue), or predicted neutral polymorphism (black). For all three panels, reported expression levels are relative to WT results and are expressed as mean ± SEM based on at least three independent experiments. (A) Total expression levels plotted as a scatterplot versus surface expression. The inset boxes illustrate which sets of mutants yield near-zero or WT-like (100%) surface expression levels. (B) Surface trafficking efficiency versus total expression level, where efficiency is defined as [(surface)mutant/(total)mutant]/[(surface)WT/(total)WT] × 100. (C) Cell surface expression levels as measured in this study versus K+ channel peak current density, as originally reported elsewhere (22). The vertical red lines indicate 65% of WT, the effective upper limit cutoff for LOF.

  • Fig. 2 Treatment of cells with a proteasome inhibitor (MG132) has modest impact on surface expression levels of the trafficking-deficient KCNQ1 variants (A) but often increases the total expression (B).

    Cells expressing WT or mutant KCNQ1 were treated with 25 μM MG132 or vehicle for 20 hours. Cells were fixed and permeabilized (this step was omitted for measuring the surface expression) and then stained with myc-tag mouse monoclonal antibody. Cells were then washed and stained with anti-mouse Alexa Fluor 647 antibody. Immunostaining of 2500 cells was quantitated by flow cytometry. Results are expressed as mean ± SEM for at least three independent experiments. Data are color-coded: LQTS mutant (red), VUS (blue), or predicted neutral polymorphism (black). Data labeled with an asterisk and a horizontal bar indicate those for which the measured KCNQ1 protein level for vehicle-treated cells was statistically different from the level measured in MG132-treated cells (P < 0.05).

  • Fig. 3 Effect on KCNQ1 trafficking of coexpression of WT KCNQ1 with mutant KCNQ1.

    (A) Total WT + mutant expression levels and (B) surface WT + mutant expression levels. HEK293 cells were transiently transfected with either 0.5 μg WT or mutant plasmid only (results on the left of each panel) or were cotransfected with both 0.25 μg WT and 0.25 μg mutant plasmids (heterozygous conditions, results presented on the right of each panel). See the legend of Fig. 1 for additional details.

  • Fig. 4 NMR spectra of WT KCNQ1 and representative mutant forms.

    1H/15N-TROSY NMR spectra (900 MHz) of the WT KCNQ1 VSD (residues 100 to 249) (A) and representative mutant forms (B to D). ppm, parts per million. The spectrum of each mutant VSD is shown in red, superimposed on the black spectrum of the WT VSD spectrum. Data were collected at 50°C for WT and mutant forms of VSD in LMPG micelles at pH 5.5. The LMPG concentration for all samples was 50 to 80 mM, and the KCNQ1 VSD concentration was 0.3 mM in all samples. (B) The spectrum from the V110I mutant, for which the only changes relative to the WT spectrum are shifts in peak positions. (C) The spectrum from the H126L mutant that is deemed to be moderately destabilized on the basis of a modest degree of line broadening for a number of peaks relative to the corresponding peaks in the WT spectrum. (D) The spectrum from the E115G mutant, which is deemed to be severely destabilized on the basis of extensive peak broadening and even disappearance of a number of peaks.

  • Fig. 5 Structural locations and key interactions involving mutation sites and/or S0 in the KCNQ1 VSD.

    (A) Three views of the VSD illustrating the locations of the five sites in S0 (red side chains: Q107, Y111, L114, E115, and P117) subject to LQTS mutations that destabilize the VSD, resulting in mistrafficking and degradation of KCNQ1. The side chains for four residues that interact with these S0 residues and that are also subject to destabilizing VUS or LQTS mutations resulting in channel LOF are shown in magenta. The open-state VSD coordinates from the cryo–electron microscopy (EM) structure of KCNQ1 [Protein Data Bank (PDB) ID: 5VMS] were used to generate this figure (23). The V110 LQTS mutation site is also located in S0, but the mutation does not appear to destabilize the protein. (B to F) Results from the MD simulation of the KCNQ1 VSD. (B) Structural model of the WT open-state human VSD in a DMPC bilayer after 500 ns of MD. The VSD is displayed in cartoon representation, with S1-S4 colored pale green and the S0 helix colored cyan. DMPC molecules are depicted as spheres and colored by atom identity: C, gray; O, red; N, blue; P, orange. (C to F) Nonbonded interactions involving sites in S0 and sites contacting S0 for which LQTS and VUS mutations were characterized in this study (see Results). Amino acid side chains are drawn as sticks. LQTS and VUS mutation sites are colored light red and blue, respectively. Residues for which mutations are neutral or have not been characterized in this work are colored gray and green, respectively. Predicted hydrogen bond interactions are indicated by black dotted lines and atoms. The nature of the nonbonded interactions involving S0 is further described in the main body of the text and in table S2.

  • Table 1 Classification of KCNQ1 variants.
    Classification*CriteriaVariants
    INormal or higher surface trafficking levels, but low peak current density.
    Dysfunctional channel.
    V110l, C122Y, L134P, A150T, A150V, T169M, K218E, I227L, and
    Q234P
    IILow surface trafficking level but normal or higher channel function for the
    minority of the population that reaches the plasma membrane.
    Q107H and P197L
    IIINormal or higher surface levels and peak current density but altered channel
    V1/2 and/or deactivation rate.
    H105L, R109L, T118S, and I132L
    IVDefective in both channel properties and surface expression levels.T104I, L131P, E160K, K196T, P197S, L236P, and L236R
    VSeverely expression- or trafficking-defective. Current is so low that channel
    properties cannot be assessed.
    Y111C, L114P, E115G, P117L, Y125D, H126L, ΔF167, R174C,
    R174H, R174L, W176R, G179S, G189A, and R195P
    VI (WT-like)Normal or higher properties in all testedV100I, A102S, T104S, H105N, H105Y, V106I, V124I, F127L,
    A128T, V129I, V133I, V135A, V135I, A149V, and V207M

    *The 32 mutants in classes I, II, IV, and V are all deemed to be LOF because they exhibit maximal channel conductance ≤65% of WT. Surface expression levels were deemed defective if they were ≤65% of WT. The four class III mutants exhibit normal maximal peak currents but altered V1/2 and/or deactivation rates and are therefore deemed dysfunctional.

    The listed mutants are color-coded to indicate their initial classifications before this work: LQTS (red), VUS (blue), or neutral/benign (black).

    H105L exhibited a hyperpolarizing shift in V1/2 for channel activation, R109L exhibited a hyperpolarizing shift in V1/2 for activation, T118S exhibited a depolarizing shift in V1/2 for activation, and I132L exhibited slowed deactivation.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/3/eaar2631/DC1

      fig. S1. Locations of the human KCNQ1 mutation examined in this work.

      fig. S2. Fluorimetric cell flow cytometry assay used to determine total and surface protein expression levels.

      fig. S3. Surface trafficking efficiency for each mutant juxtaposed with KCNQ1 peak current density.

      fig. S4. Effect of coexpression of KCNE1 with KCNQ1 on total surface expression levels of KCNQ1.

      fig. S5. 1H/15N-TROSY NMR spectra (900 MHz) of KCNQ1 mutants (red) superimposed on the spectrum of WT KCNQ1 (black).

      fig. S6. Results from MD trajectories.

      fig. S7. Hydrogen bond interactions of the KCNQ1 VSD with solvent are observed during MD simulation.

      fig. S8. Correlation plot of residue motions calculated from KCNQ1 VSD MD simulations.

      table S1. Functional and trafficking results for 51 human KCNQ1 mutants.

      table S2. List of nonbonded interactions of LQTS1 and VUS mutation sites in S0 and S0-contacting regions observed during MD simulation of the KCNQ1 VSD.

    • Supplementary Materials

      This PDF file includes:

      • fig. S1. Locations of the human KCNQ1 mutation examined in this work.
      • fig. S2. Fluorimetric cell flow cytometry assay used to determine total and surface protein expression levels.
      • fig. S3. Surface trafficking efficiency for each mutant juxtaposed with KCNQ1 peak current density.
      • fig. S4. Effect of coexpression of KCNE1 with KCNQ1 on total surface expression levels of KCNQ1.
      • fig. S5. 1H/15N-TROSY NMR spectra (900 MHz) of KCNQ1 mutants (red) superimposed on the spectrum of WT KCNQ1 (black).
      • fig. S6. Results from MD trajectories.
      • fig. S7. Hydrogen bond interactions of the KCNQ1 VSD with solvent are observed during MD simulation.
      • fig. S8. Correlation plot of residue motions calculated from KCNQ1 VSD MD simulations.
      • table S1. Functional and trafficking results for 51 human KCNQ1 mutants.
      • table S2. List of nonbonded interactions of LQTS1 and VUS mutation sites in S0 and S0-contacting regions observed during MD simulation of the KCNQ1 VSD.

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