Photo-crosslinking hERG channels causes a U.V.-driven, state-dependent disruption of kinetics and voltage dependence of activation

Human ether-a-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. Characteristic slow closing (deactivation) in hERG is regulated by direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). We aim to understand how the PAS domain that is distal to the pore rearranges during gating to allosterically regulate the channel pore (and ion flux). 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 double radical capable of forming covalent cross-links with C-H bond-containing groups, enabling selective and potent U.V.-driven photoinactivation of ion channel dynamics. Here we incorporate BZF directly into the hERG potassium channel PAS domain at three locations (G47, F48, and E50) using TAG codon suppression technology. hERG channels with BZF incorporated into the PAS domain (hERG-BZF) showed a significant change in the biophysical properties of the channel. hERG-G47BZF activated slowly when irradiated in the closed state (-100mV) but deactivated quickly when irradiated in both the open (0mV) and closed state. hERG-F48BZF channels showed a state independent and U.V. dose-dependent change in channel activation (slowing down) and channel deactivation (speeding up), as well as a marked change (right-shift) in the voltage-dependence of conductance. When irradiated at -100 mV hERG-E50BZF showed a state dependent and U.V. dose-dependent change in a channel activation (slowing down) and deactivation (speeding up) of channel deactivation, as well as a marked change (right-shift) in the voltage-dependence of conductance that occurred only when the channel was irradiated in the closed state (-100mV). This approach demonstrated that direct photo-crosslinking of the PAS domain in hERG channels causes a measurable change in biophysical parameters and more broadly stabilized the closed state of the channel. We propose that altered channel gating is as a direct result of reduced dynamic motions in the PAS domain of hERG due to photo-chemical crosslinking.