Using the photo-crosslinkable non-canonical amino acid BZF to capture U.V.-driven, state-dependent disruption of kinetics and voltage dependence of activation in hERG potassium channels.

Abstract

Human ether-à-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. hERG deactivation is regulated by interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). Now, we aim to understand the how the conformational dynamics and accessible landscape of the hERG PAS domain contributes to channel kinetics and voltage dependence. To achieve this, we utilized the non-canonical amino acid 4-Benzoyl-L-phenylalanine (BZF) which is a photo-activatable cross-linkable probe, that when irradiated with ultraviolet (U.V.) light forms a covalent cross-link with C-H bond-containing groups, enabling selective and potent U.V.-driven state-dependent photoinactivation modification of ion channel dynamics. This is because BZF can form bonds to nearby atoms via cross-linkable reactivity reducing degrees of freedom within a domain of the channel. Here we incorporate BZF directly into the hERG potassium channel using TAG codon suppression that, upon U.V exposure, shows a large change in the biophysical properties of the channel when irradiated at −100 mV when the channels are closed. When compared to wild-type hERG1a, hERG1a-BZF shows a U.V. dose-dependent change (speeding up) of channel deactivation. Additionally, hERG1a-BZF exhibits a marked change (right-shift) in the voltage-dependence of activation when irradiated at −100 mV when the channels are closed. These effects are not observed when the hERG1a-BZF channel is irradiated in at 0 mV when the channels are open. This approach demonstrates that direct photo-crosslinking of KCNH hERG channels causes a measurable change in biophysical parameters and this effect is state-dependent (occurring here in the closed state but not the open state). We propose that altered channel gating is as a direct result of reduced dynamic motions in the hERG channel due to photo-chemical crosslinking.