Structure and physiological function of the human KCNQ1 channel voltage sensor intermediate state

  1. Keenan C Taylor
  2. Po Wei Kang
  3. Panpan Hou
  4. Nien-Du Yang
  5. Georg Kuenze
  6. Jarrod A Smith
  7. Jingyi Shi
  8. Hui Huang
  9. Kelli McFarland White
  10. Dungeng Peng
  11. Alfred L George
  12. Jens Meiler
  13. Robert L McFeeters
  14. Jianmin Cui  Is a corresponding author
  15. Charles R Sanders  Is a corresponding author
  1. Department of Biochemistry, Vanderbilt University, United States
  2. Center for Structural Biology, Vanderbilt University, United States
  3. Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, and Cardiac Bioelectricity, and Arrhythmia Center, Washington University in St. Louis, United States
  4. Departments of Chemistry and Pharmacology, Vanderbilt University, United States
  5. Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, United States
  6. Department of Pharmacology, Northwestern University Feinberg School of Medicine, United States
  7. Department of Bioinformatics, Vanderbilt University Medical Center, United States
  8. Department of Chemistry, University of Alabama in Huntsville, United States
  9. Department of Medicine, Vanderbilt University Medical Center, United States
11 figures, 2 tables and 1 additional file

Figures

Figure 1 with 3 supplements
NMR spectra of the human KCNQ1 voltage-sensor domain.

(A) 1H-15N TROSY-HSQC spectrum recorded at 900 MHz of 2H,13C,15N-Q1-VSD in LMPG micelles. Backbone amide peaks for 140 out of 147 non-proline residues (95%) have been assigned. Only a single set of …

Figure 1—figure supplement 1
Long range PREs are consistent with the expected KCNQ1 VSD topology in LMPG micelles.

The residue positions of the spin-labeled cysteines for each sample are : (A) 144-SL (B) 155-SL (C) 214-SL (D) 224-Sl (E) 121-SL (F) 177-Sl (G) 180-SL (H) 238-SL.

Figure 1—figure supplement 2
TALOS-N secondary structure analysis of backbone chemical shifts (top panel) and (bottom panels) deviations of the (Osteen et al., 2012) Cα and carbonyl 13C’ chemical shifts from random coil values.

The chemical shift data for the KCNQ1 VSD been deposited in BioMagResBank (BMRB ID 30517).

Figure 1—figure supplement 3
Examples of NOE measurements.

Example strip plots showing long-range methyl-methyl and methyl-backbone Hα proton NOEs (red arrows) taken from the methyl-optimized 3D 13C-edited NOESY recorded on a 13C,15N-labeled sample in …

Figure 2 with 1 supplement
Structure of the KCNQ1 VSD.

(A) The human KCNQ1 VSD NMR-determined ensemble after molecular dynamics refinement in a hydrated DMPC bilayer (PDB ID 6MIE, see statistics in Table 1 and also Figure 2—figure supplement 1) (B) …

Figure 2—figure supplement 1
KCNQ1-VSD XplorNIH-determined structural ensemble, before and after molecular dynamics refinement.

(A) The ensemble is comprised of the top 15 lowest energy structures out of 150 conformations generated by NMR data-restrained XPLOR-NIH calculations (no bilayer). The transmembrane helices S1, S2, …

Figure 2—figure supplement 1—source data 1

Text file with coordinates (PDB format) of the XPLOR-NIH structure ensemble for the KCNQ1-VSD prior to molecular dynamics.

https://cdn.elifesciences.org/articles/53901/elife-53901-fig2-figsupp1-data1-v2.txt
Figure 2—figure supplement 1—source data 2

Excel file with numerical data used for Figure 2—figure supplement 1C, top panel.

https://cdn.elifesciences.org/articles/53901/elife-53901-fig2-figsupp1-data2-v2.xlsx
Figure 2—figure supplement 1—source data 3

Excel file with numerical data used for Figure 2—figure supplement 1C, bottom panel.

https://cdn.elifesciences.org/articles/53901/elife-53901-fig2-figsupp1-data3-v2.xlsx
Figure 2—figure supplement 1—source data 4

Excel file with numerical data used for Figure 2—figure supplement 1D, top panel.

https://cdn.elifesciences.org/articles/53901/elife-53901-fig2-figsupp1-data4-v2.xlsx
Comparison of intermediate and activated KCNQ1 VSD conformations.

(A) Intermediate conformation of human KCNQ1 VSD (1st structure in the PDB 6MIE ensemble). (B) Activated conformation of the Xenopus KCNQ1 VSD (PDB 5VMS) (Sun and MacKinnon, 2017). In both panels, …

S2—S4 salt bridges/hydrogen bonds in the NMR and cryo-EM structures of the KCNQ1 VSD.

(A) Intermediate state conformation of human KCNQ1 VSD (1st structure in the PDB 6MIE ensemble). Of particular note are the ionic interactions of E1-R2 and E2-R4, as well as, the close packing of Q3 …

Figure 5 with 1 supplement
Schematics and electrophysiology data validating the intermediate and activated KCNQ1 VSD functional states utilizing auxiliary subunit KCNE1 regulation as a probe.

Amino acid residue nomenclature: E2 = E170, R4 = R237, F0 = F167, Q3 = Q234, and H5 = H240. Numbering corresponds to the human KCNQ1 sequence. All error bars are ± SEM. All horizontal scale bars …

Figure 5—figure supplement 1
Electrophysiology results for KCNQ1 F0R single and double mutants with and without KCNE1 co-expression, and with XE991 exposure.

F0 = F167; Q3 = Q234; R4 = R237; H5 = H240. Residue numbers correspond to human KCNQ1. All error bars are ± SEM. (A) Top: Current recordings of KCNQ1 F0R single mutant with and without KCNE1 …

Figure 5—figure supplement 1—source code 1

MATLAB script that takes in tail current data obtained from PatchMaster program (see Key Resources Table), fits the data with a Boltzmann equation, and outputs the best fit parameters.

https://cdn.elifesciences.org/articles/53901/elife-53901-fig5-figsupp1-code1-v2.m
Figure 5—figure supplement 1—source data 1

Excel file with numerical data used for Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/53901/elife-53901-fig5-figsupp1-data1-v2.xlsx
Electrophysiology validating the intermediate and activated KCNQ1 VSD functional states utilizing XE991 pharmacology as a probe.

Amino acid residue nomenclature and numbering and error bars are similar as in Figure 5. (A) Table outlining 5 µM XE991 effect on KCNQ1 IO and AO state currents based on prior studies (Zaydman et …

KCNE3 shifts the voltage dependence of the intermediate-open state to render its conductance relevant under physiological conditions.

(A) Voltage-clamp fluorometry recordings for pseudo-WT (C214A/G219C/C331A) KCNQ1 (left), KCNQ1+KCNE1 (middle), and KCNQ1+KCNE3 (right). Current (black) and fluorescence (blue) were recorded with …

Author response image 1
XplorNIH NMR ensemble prior to MD refinement displaying the orientation of the key sidechain residues.

The S0 and S1 helices are not shown to improve clarity.

Author response image 2
XplorNIH ensemble before (left) and after (middle) RDC refinement.

The RDC refined ensemble is colored gray and the NOE/PRE derived ensemble is colored teal.

Author response image 3
5 µM XE991 inhibition of KCNQ1, KCNQ1+KCNE1, and KCNQ1+KCNE3 channels at 300 ms activation duration.

% inhibition was calculated with 1 – (IXE991 / IControl) at 300 ms after the test pulse started.

Author response image 4
VCF results of E1R/R2E (A) and R0R/H5E (B).

Tables

Table 1
KCNQ1 VSD NMR structure statistics.
Structure restraintsXPLOR-NIH*PDB 6MIE†
Total NOE958958
 Inter-residue:
Sequential ( | i - j | = 1 )559559
Medium-range ( 1 < | i - j | < 5 )366366
Long-range ( | i - j | ≥ 5 )3333
Hydrogen bonds5555
Paramagnetic relaxation enhancement403403
Dihedral angle
ϕ9797
φ9797
Residual dipolar couplings (DHN)5454
Structure statistics
Ensemble r.m.s.d. (residues 120–152, 160–239)
Backbone heavy atoms (Å)1.410.96
All heavy atoms (Å)2.331.72
Transmembrane r.m.s.d. (residues 120–142, 160–179, 198–215, 219–239)
Backbone heavy atoms (Å)0.870.97
r.m.s.d. from experimental restraints
Distances (Å)0.068 ± 0.0050.150 ± 0.019
Dihedral angles (°)1.0 ± 0.210.3 ± 4.2
Residual dipolar coupling (Hz)0.92 ± 0.213.1 ± 0.6
r.m.s.d. from idealized geometry
Bond lengths (Å)0.003 ± 0.0010.005 ± 0.001
Bond angles (°)0.44 ± 0.011.71 ± 0.01
Ramachandran plot (residues 101–152, 160-239)
Most favorable (%)89.3 ± 2.089.8 ± 2.8
Additionally allowed (%)10.0 ± 2.28.8 ± 2.3
Generously allowed (%)0.4 ± 0.80.7 ± 0.6
Disallowed (%)0.3 ± 0.80.6 ± 0.7
  1. *'XPLOR-NIH’ describes the statistics for the XPLOR-NIH structure ensemble generated using experimental restraints, prior to the rMD phase of the calculations.

    †'MD’ describes the statistics for the structure ensemble (PDB ID: 6MIE) (see Materials and methods).

  2. ‡Procheck NMR.

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (human)KCNQ1HUGO Gene Nomenclature Committee (HGNC)Gene ID: 3784;
HGNC:6294
Gene (human)KCNE1HUGO Gene Nomenclature Committee (HGNC)Gene ID: 3753;
HGNC:6240
Gene (human)KCNE3HUGO Gene Nomenclature Committee (HGNC)Gene ID: 10008;
HGNC:6243
Strain, strain background (Escherichia coli)CT19 transaminase deficient strainDr. David Waugh, US National Cancer Institute
PMID: 8914274
Used for special isotopic labeling of the KCNQ1 VSD for use in NMR studies.
Strain, strain background (Escherichia coli)Rosetta/
C43(DE3)
Sigma-AldrichCatalog number 70954Used for uniform isotopic labeling of KCNQ1 and amino acid-specific labeling for NMR.
Recombinant DNA reagentpET16b expression plasmid encoding tagged human KCNQ1-
(100-249)
PMID: 24606221Used to express the human KCNQ1 VSD for NMR structural studies
Biological sample (include species here)Xenopus oocytes
(Xenopus laevis, female)
This paperRRID:XEP_XlaXenopus laevis purchased from Nasco, Fort Atkinson, WI
Recombinant DNA reagentpcDNA3.1 encoding human KCNQ1 or KCNE1This paperRRID:Addgene_111452For site-directed mutagenesis
Commercial assay or kitmMessage T7 polymerase kitApplied Biosystems-Thermo Fisher ScientificAM1344
Chemical compound, drugXE991Millipore SigmaCAS #:
122955-42-4
Chemical compound, drugChromanol 293BMillipore SigmaCAS #:
163163-23-3
Chemical compound, drugAlexa Fluor488 C5 maleimideMolecular Probes, Eugene, ORCatalog #: A10254
SoftwareTopspin 3.2Bruker (Scientific Instruments Company)RRID:SCR_014227NMR data collection and processing.
SoftwareMDD and qMDD interfaceURL: mddnmr.spektrino.com/
PMID: 21161328
NMR data processing
SoftwareNMRFAM-SPARKYPMID: 25505092
URL: https://nmrfam.wisc.edu/nmrfam-sparky-distribution/
NMR data analysis and
resonance assignments.
SoftwareTALOS-NPMID:25502373
URL: spin.niddk.nih.gov/bax/software/TALOS-N/
Determination of secondary structure from NMR chemical shift data.
SoftwareCHARMM-GUIPMID: 25130509
URL: www.charmm-gui.org/
Preparation of starting structures of the KCNQ1 VSD in lipid bilayers for MD restrained MD simulations.
SoftwareXPLOR-NIHPMID: 28766807
URL: https://nmr.cit.nih.gov/xplor-nih/
Structure calculations using NMR data restraints
SoftwareGPU-accelerated AMBER 16URL: https://ambermd.org/
PMID: 16200636
Program for execution of MD simulations.
SoftwareLipid 17 AMBERPMID: 24803855
URL: https://ambermd.org/AmberModels.php
Force field used for MD simulations.
SoftwareCPPTRAJPMID: 26583988
URL:https://amber-md.github.io/
Analysis of MD trajectoriesfollowing simulations.
SoftwarePatchMasterHEKARRID:SCR_000034Electrophysiology data collection
SoftwareIGORWavemetrics, Lake Oswego, ORRRID:SCR_000325Electrophysiology data analysis
SoftwareClampfitAxon Instruments, Sunnyvale, CARRID:SCR_011323Electrophysiology data analysis
SoftwareSigmaplotSPSS, San Jose, CARRID:SCR_003210Electrophysiology data analysis and visualization
SoftwareMATLABMathWorks, MARRID:SCR_001622Electrophysiology data analysis
Sequence-based reagentFor site-directed mutagenesisThis paperPCR primersPCR primers seq for mutations made in this study (each mutation utilizedtwo primers: b and c).
E170R-b:cacgtacCTGgtcccgaagaacaccac;
E170R-c: cgggacCAGgtacgtggtccgcctc;
R237E-b: gcatcTCcaggatctgcaggaag;
R237E-c:cagatcctgGAgatgctacacgtcgac
F167R-b: ccgtcccgCGgaacaccaccagcac;
F167R-c: gtgttcCGcgggacggagtacg
Q234E-b:ggatctCcaggaagcggatgccc;
Q234E-c: catccgcttcctgGagatcctgaggatgcta
H240E-b: cggtcgacCTCtagcatcctcaggatc
H240E-c: gctaGAGgtcgaccgccaggg
D202N-b: cgatgaggtTAatgatggaaatgggcttc
D202N-c: ccatcatTAacctcatcgtggtcgtg
F351A-b: ggcaGCGcccgagccaagaatcc
F351A-c: gctcgggCGCtgccctgaaggtgcag
C214A-b: cttggaTcccacCGCgaggaccacca
C214A-c: cGCGgtgggAtccaaggggcaggtg
G219C-b: cctgaCacttggaTcccacCGC
G219C-c: ggAtccaagtGtcaggtgtttgccacg
C331A-b: gacagagaaTGCggaggcgatggtcttc
C331A-c: ctccGCAttctctgtctttgccatc

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