Dynamics of the PAS and CNBHD Domain Interaction Probed with a Fluorescent Noncanonical Amino Acid (L-ANAP) in hERG Potassium Channels

Abstract

The voltage-gated potassium channel hERG 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 PAS domain and the C-terminal CNBHD. The PAS-CNBHD interaction is sensitive to point mutations at the domain interface in hERG but less is known about potential dynamic rearrangements of the PAS domain relative to the CNBHD and the rest of the channel. Here, we report that hERG channels showed FRET that is sensitive to voltage and potassium-induced cell depolarization, consistent with a PAS-CNBHD rearrangement. We took advantage of a fluorescent noncanonical amino acid (L-ANAP) and a metal ion bound to a dihistidine motif that can be used to monitor small structural rearrangements within ion channels using patch-clamp fluorometry (PCF) and transition metal FRET (tmFRET). Using amber stop codon suppression technology, we show that amber stop codon-containing mutants in the PAS domain incorporate L-ANAPs as shown by robust currents measured with two-electrode voltage-clamp. Likewise, the addition of dihistidine mutations in the CNBHD also result in functional channels. We performed PCF recordings of excised patches containing L-ANAP-incorporated channels and show that hERG channels are stable in excised patches with robust L-ANAP and Citrine fluorescence that is linearly correlated, suggesting that L-ANAP has been site-specifically incorporated into the channel. We then exposed the internal face of the excised patch to transition metal ions, which bind to the dihistidine motif to quench nearby L-ANAP in a distance- dependent matter. We report a voltage-dependent change in L-ANAP fluorescence in the presence of metal ions, demonstrating a rearrangement of the PAS and CNBHD during slow deactivation of the channel.