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Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating - PubMed

  • ️Fri Jan 01 2021

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating

Carol A Harley et al. Proc Natl Acad Sci U S A. 2021.

Abstract

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Keywords: CNBh domain; FRET; LQTS; PAS domain; scFv antibodies.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

Epitope of scFv2.12. (A) Cartoon representing the intensity decays across the PAS residues monitored by NMR spectroscopy upon scFv2.12 titration. Circle width and color define the number of titrant equivalents at which a given residue is titrated out, as indicated. Epitope and secondary structure elements are labeled. (B) Two views of PAS domain structure with the number of equivalents of titrant required to titrate out the residue (as determined in A) color coded on the surface of the domain in a gray–white–red color scale (gray, 1:0.65; red, 1:0.25). Dashed circle shows the cluster of PAS residues that are more sensitive to scFv2.12. Views are related by 180° rotation. (C) Cartoon of PAS domain structure with residues in cluster shown in B as spheres. (D) Residues in cluster were individually mutated to alanine, and mutant proteins evaluated for binding to scFv2.12 by ELISA. Plates were coated with scFv2.12 SNAPHis6 protein and binding of wild-type GST-PAS (WT) or GST-PAS protein with single point mutations in the PAS domain (N33A, R35A, V36A, E37A, N38A, and I42A) evaluated. Error bars denote mean ± SD and n = 12. Reference

SI Appendix, Fig. S1

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Fig. 2.
Fig. 2.

Mutations in scFv2.12 epitope destabilize PAS–CNBh functional interactions. (A) Surface representation of hERG channel cryo-EM structure and zoom view (Right) with scFv2.10 and scFv2.12 epitopes mapped (red surfaces) onto a single subunit of the channel. The epitope of scFv2.12 is at the interface between a CNBh domain (in yellow) and its PAS domain partner. Lines represent potential limits of membrane bilayer. (B) Scaled tail currents of wild-type (WT) and N33A, V36G, V36S, E37S, R35A, and I42A mutant channels and of WT channels coexpressed with scFv2.12 antibody. Tail currents were evoked at −120 mV after a step to +60 mV at room temperature. Dotted line indicates zero current level. (C) Fast component of deactivation in control, mutant channels, and WT hERG1a channels coexpressed with the scFv2.12 antibody (#, P < 0.05; NS, not significant). Data are mean ± SEM. Reference

SI Appendix, Fig. S2

.

Fig. 3.
Fig. 3.

Antibody affects deactivation exclusively via interactions with defined epitope. (A) Scaled tail currents of wild-type (WT) and mutant channels R35A, E37A, and R35A/E37A with and without coexpressed scFv2.12 antibody. Tail currents were evoked at −120 mV after a step to +60 mV at room temperature. Dotted line indicates zero current level. (B) Fast component of deactivation in WT and mutant channels with and without coexpressed scFv2.12 antibody (#, P < 0.05; NS, not significant). Data are mean ± SEM.

Fig. 4.
Fig. 4.

scFv2.12 and CNBh domain compete for binding to the PAS domain. (A) Competition ELISA evaluating wild-type PAS domain (1 μM) binding to scFv2.10 or scFv2.12 (coated on the plate) in the presence of increasing concentration of CNBh domain protein. Error bars denote mean ± SD and n = 8. (B) Plot of relative FRET efficiency in cells coexpressing PAS-CFP (donor) and ΔPAS-hERG Citrine (acceptor) in the absence (control) and presence of scFv2.12, administered through the patch pipette at room temperature. Error bars denote mean ± SEM.

Fig. 5.
Fig. 5.

scFv2.12 is a gating sensor. (A) Schematic diagram of donor scFv2.12(4aa)mCerulean and acceptor hERGmVenus (hERGV) or ΔPAS-hERGmVenus (ΔhERGV) constructs used for cotransfection experiments in HEK293 cells. (B) Dot plot of relative FRET efficiency values from individual cells cotransfected with scFv2.12(4aa)mCerulean and hERGV or ΔhERGV channels subjected to 5 mM K+ (LOW K+) or 150 mM K+ (HIGH K+) at 37 °C. Error bars denote mean ± SEM and n ≥ 10. Statistical analysis with unpaired t test and P ≤ 0.001 (***) and P > 0.05 (n.s). Reference

SI Appendix, Figs. S3–S5 and Table S1

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Fig. 6.
Fig. 6.

PAS–CNBh interaction is state dependent. (A) Cartoon representation of two models proposed for interpretation of the state-dependent FRET data collected with scFv2.12. The altered PAS–CNBh assembly is depicted as a dissociated PAS domain for clarity of representation. scFv2.12 fused to mCerulean is represented by black and cyan ovals; hERG channel fused to mVenus is shown in gray and yellow. Closed gate is indicated by cross, open gate by white oval. hERG cytoplasmic domains are indicated in a single subunit. Arrow and FRET label indicate spatial conditions where FRET occurs. Double arrow indicates that the conformation of hERG changes with K+ conditions. (B) Schematic diagram of donor scFv2.12(4aa)mCerulean and acceptor R56Q hERGmVenus (R56Q hERGV) or wild-type hERGmVenus (hERGV) constructs used for cotransfection experiments in HEK293 cells. (C) Dot plot of relative FRET efficiency values from individual cells cotransfected with scFv2.12(4aa)mCerulean and hERGmVenus (hERGV) or R56Q hERG mVenus (R56Q hERGV) channels subjected to 5 mM K+ (LOW K+) or 150 mM K+ (HIGH K+) at 37 °C. Error bars denote mean ± SEM and n ≥ 10. Statistical analysis with unpaired t test and P ≤ 0.001 (***) and P > 0.05 (n.s). Reference

SI Appendix, Table S1

.

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