Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating
Carol A Harley1,2, Ganeko Bernardo-Seisdedos3, Whitney A Stevens-Sostre4
1Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), Porto 4200-135, Portugal.
Insights
The human Ether-à-go-go-Related Gene (hERG) channel
Area of Science:
- Cardiovascular Physiology
- Molecular Biology
- Ion Channel Biophysics
Background:
- The human Ether-à-go-go-Related Gene (hERG) K+ channel is vital for cardiac repolarization.
- Dysfunction of hERG channels can lead to long QT syndrome and fatal arrhythmias.
- The cytosolic Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains are key structural features, but their gating role is unknown.
Purpose of the Study:
- To investigate the molecular role of the PAS and CNBh domains in hERG channel gating.
- To determine how scFv2.12 antibody binding affects the PAS-CNBh interaction.
- To elucidate the structural rearrangements of the PAS/CNBh assembly during channel gating.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy and mutagenesis to map antibody epitopes.
- In vitro and cell-based assays using fluorescently labeled antibodies.
- Förster Resonance Energy Transfer (FRET) to detect conformational changes.
- Site-directed mutagenesis (R56Q) to disrupt PAS-CNBh interaction.
Main Results:
- The scFv2.12 epitope overlaps the PAS/CNBh domain interface, and its binding prevents PAS-CNBh interaction.
- scFv2.12 binding to full-length hERG channels is state-dependent, detected only when the channel gate is open.
- The state-dependent FRET signal is abolished by the R56Q mutation, which destabilizes the PAS-CNBh interaction.
Conclusions:
- hERG channel opening is associated with significant structural changes in the PAS/CNBh assembly.
- These changes likely involve the dissociation of the PAS domain from the CNBh domain.
- Antibody binding can reveal dynamic conformational states of the hERG channel's cytosolic domains.
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.
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