An intracellular hydrophobic nexus is critical for slow deactivation in hERG channels

Slow deactivation is a critical biophysical and physiological property of voltage-gated K+ channels encoded by the human Ether-a-go-go-Related Gene (hERG). hERG channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (residues 26-136) and an N-terminal PAS-cap that is further subdivided into an initial unstructured segment (residues 1-12) and an amphipathic -helical region (residues 13-25). Disruption of interactions within the PAS-CNBhD complex accelerates deactivation kinetics and impairs the slow repolarization of the cardiac action potential. Similarly, deletion of the PAS-cap unstructured segment, modeled as a "foot-in-the-door," accelerates channel closing. Here, we tested the hypothesis that a three-dimensional hydrophobic nexus, comprising residues in the PAS-cap -helix, the globular PAS, and CNBh domains, plays a crucial role in slow deactivation. Utilizing structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we provide evidence that altering hydrophobicity at this interface accelerates deactivation kinetics and causes a rearrangement in the network of residues comprising the hydrophobic nexus. Moreover, the PAS-cap unstructured segment disengages from the gating machinery. We propose that the nexus hydrophobicity positions the PAS-cap -helix and promotes engagement of the upstream PAS-cap unstructured segment with the gating machinery, resulting in slow channel closing. Interestingly, several long QT syndrome disease mutations lie at this interface, underscoring the importance of the hydrophobic nexus and the novel role of the PAS-cap -helix in hERG gating.