Dynamics of the EAG Domain and Cyclic Nucleotide-Binding Homology Domain Interaction Probed with a Fluorescent Noncanonical Amino Acid (L-ANAP) in hERG Potassium Channels

Abstract

The voltage-gated potassium channel hERG (KCNH2, Kv11.1) plays a critical role in cardiac repolarization and is characterized by unusually slow deactivation kinetics. Slow deactivation in hERG channels is regulated by a direct interaction between the N-terminal eag domain and the C-terminal CNBHD. The eag-CNBHD interaction is sensitive to point mutations at the domain interface in hERG but less is known about potential dynamic rearrangements of the eag domain relative to the CNBHD and the rest of the channel. Here, we report that hERG channels formed from eag-CFP domains and Δeag-Citrine channels showed FRET (Förster resonance energy transfer) that is sensitive to potassium-induced cell depolarization, consistent with an eag-CNBHD rearrangement. To examine this in more detail, we took advantage of a fluorescent noncanonical amino acid (L-ANAP) and a metal ion bound to a dishistidine motif that can be used to monitor small structural rearrangements within ion channels using patch-clamp fluorometry (PCF) and transition metal FRET. We found that amber stop codon-containing mutants N33X, E50X, and C64X in the eag domain incorporate L-ANAPs as shown by robust currents measured with two-electrode voltage-clamp. Likewise, the addition of dihistidine mutations in the CNBHD (N33X_E788H,L790H, E50X_S855H,H851 and C64X_V796H,I798H) also result in functional channels. The voltage dependence of these channels was comparable to control, indicating incorporation of L-ANAP and introduction of histidines did not measurably alter channel gating. We then performed PCF recordings of excised patches with L-ANAP incorporated hERG channels and found that hERG channels are stable in excised patches and that hERG E50X_S855H channels showed robust L-ANAP and Citrine fluorescence. The above techniques will determine how the eag-CNBHD interaction dynamically regulates conformational changes and ionic current in hERG.