Photoinhibition of the hERG potassium channel PAS domain by ultraviolet light speeds channel closing

Abstract

hERG potassium channels are critical for cardiac excitability. hERG channels have a Per-Arnt-Sim (PAS) domain at their N-terminus, and here, we examined the mechanism for PAS domain regulation of channel opening and closing (gating). We used TAG codon suppression to incorporate the noncanonical amino acid 4-benzoyl-L-phenylalanine (BZF), which is capable of forming covalent cross-links after photoactivation by ultraviolet (UV) light, at three locations (G47, F48, and E50) in the PAS domain. We found that hERG-G47BZF channels had faster closing (deactivation) when irradiated in the open state (at 0 mV) but showed no measurable changes when irradiated in the closed state (at -100 mV). hERG-F48BZF channels had slower activation, faster deactivation, and a marked rightward shift in the voltage dependence of activation when irradiated in the open (at 0 mV) or closed (at -100 mV) state. hERG-E50BZF channels had no measurable changes when irradiated in the open state (at 0 mV) but had slower activation, faster deactivation, and a rightward shift in the voltage dependence of activation when irradiated in the closed state (at -100mV), indicating that hERG-E50BZF had a state-dependent difference in UV photoactivation, which we interpret to mean that PAS underwent molecular motions between the open and closed states. Moreover, we propose that UV-dependent biophysical changes in hERG-G47BZF, F48BZF, and E50BZF were the direct result of photochemical cross-linking that reduced dynamic motions in the PAS domain and broadly stabilized the closed state relative to the open state of the channel.

Figure 1

Incorporation of the noncanonical amino acid BZF into the PAS domain of hERG K channels. (A) Schematic of two (of four) hERG subunits showing key functional domains including the N-terminal (Per-Arnt-Sim) PAS-Cap domain (light green), PAS domain and alpha A helix (green), N-linker region (black), voltage sensor domain S1–S3 helices (gray) and S4 helix (dark gray), pore domain composed of the S5 helix, loop, and S6 helix (black), C-linker (blue), cyclic nucleotide-binding homology domain (CNBHD) (purple), intrinsic ligand (orange), and C-terminal region (black). (B) hERG tetramer modeled from structurally determined coordinates with similar color-coding of functional domains as in (A). (PDB: 5VA2). (C) Phylogenetic tree of hERG and related ion channels. (D) For amber codon suppression, a Xenopus oocyte (large circle) was injected in the nucleus with a plasmid that encodes both the transfer RNA (tRNA^BZF) and RNA synthetase (RS^BZF), also known as the orthogonal set, which binds and then charges tRNA with BZF. The charged tRNA^BZF is used by the ribosome (gray schematic) to read through a genetically modified version of hERG mRNA containing a TAG stop (amber) codon and inserts BZF into the nascent peptide that is translocated to the membrane as a full-length channel (purple) with BZF incorporated (red circle). (E) BZF incorporated into a protein (top) will be part of the primary peptide chain with the amino group forming a peptide bond to the upstream endogenous amino acid (R₁) and the carboxyl group bound to the endogenous downstream amino acid (R₂) and harbors the functional group (benzophenone) attached to Cα. When exposed to UV light, BZF forms a double radical capable of eliminating water and forming a new carbon-carbon bond depicted here with a protein, where R₃ is the moiety disrupted by new bond formation and is dependent on localized chemistry. (F) Structure of PAS (green) and CNBHD (purple) from adjacent hERG subunits (PDB: 5VA2) and the locations of residues G47, F48, and E50 in the PAS alpha A helix with side chains shown as sticks. (G) Plot of peak current measured at 40 mV from two-electrode voltage-clamp recordings from oocytes after expression of hERG (as a negative control) or hERG-G47TAG, hERG-F48TAG, and hERG-E50TAG mutants with the orthogonal set and either without BZF (+/−) (black hollow symbols) or with BZF (+/+) (black solid symbols) added to the bath solution. n ≥ 3, ^∗∗p ≤ 0.01.

Figure 2

Currents and steady-state activation of hERG and hERG-BZF channels before and after UV irradiation in the closed state. Family of currents from (A and B) hERG, (D and E) hERG G47BZF, (G and H) hERG F48BZF, and (J and K) hERG E50BZF channels, recorded before (0 s) UV irradiation (black traces) or after 15 s UV irradiation (blue traces). UV light was applied in the closed state (−100 mV) in all experiments. Voltage protocol (red traces, inset) was 20-mV steps from a holding voltage of −100 mV. Scale bars are indicated for each current family. Conductance-voltage (G-V) plots for (C) hERG, (F) hERG G47BZF, (I) hERG F48BZF, and (L) hERG E50BZF, before (0 s) or after UV irradiation for durations of 0, 1, 3, 7 or 15 s. A right-shift in the GV (black arrow) was measured for hERG F48BZF and hERG E50BZF. (M) Scatter plot of the midpoint of the GV curves before (black symbols) or after (blue symbols) 15 s of UV irradiation. n = 3 for each experiment. ns denotes p > 0.05, ^∗∗ denotes p ≤ 0.01, ^∗∗∗ denotes p ≤ 0.001.

Figure 3

Currents and steady-state activation of hERG and hERG-BZF channels before and after UV irradiation in the open state. Family of currents from (A and B) hERG, (D and E) hERG-G47BZF, (G and H) hERG-F48BZF, and (J and K) hERG-E50BZF channels, recorded before (0 s) UV irradiation (black traces) or after (blue traces) 15 s of UV irradiation. UV light was applied in the open state (0 mV) in all experiments. After UV irradiation at 0 mV, currents were elicited by a voltage protocol (red traces, inset) with 20-mV steps from a holding voltage of −100 mV. Scale bars are indicated for each family of currents. Conductance-voltage (G-V) plots for (C) hERG, (F) hERG-G47BZF, (I) hERG-F48BZF, and (L) hERG-E50BZF, before (0 s) or after UV irradiation for durations of 0, 1, 3, 7 or 15 s. A right-shift in the GV (black arrow) was measured for hERG-F48BZF. (M) Scatter plot of the midpoint of the G-V curves before (black symbols) or after (blue symbols) 15 s of UV irradiation. n = 3 for each experiment. ns denotes p > 0.05, ^∗ denotes p ≤ 0.05.

Figure 4

hERG and hERG-BZF channel tail currents report the time course of channel deactivation before and after UV irradiation. Normalized tail currents from (A and B) hERG, (C and D) hERG-G47BZF, (E and F) hERG-F48BZF, or (G and H) hERG-E50BZF before (black traces) or after 15 s of UV irradiation (blue traces). UV light was applied when channels were held in closed state at −100 mV or in the open state at 0 mV, as indicated. The scale bar represents 100 ms, and the dashed line indicates zero current level. Inset (red trace) depicts voltage pulse. Scatter plots of the time constant of deactivation (tau fast) before (black symbols) or after (blue symbols)15 s of UV irradiation applied at (I) −100 mV or (J) 0 mV. n = 3, ns denotes p > 0.05, ^∗∗ denotes p ≤ 0.01, ^∗∗∗ denotes p ≤ 0.005, ^∗∗∗∗ denotes p ≤ 0.005.

Figure 5

Current traces indicate time course of activation before and after UV irradiation. Normalized currents recorded from (A and B) hERG, (C and D) hERG-G47BZF, (E and F) hERG- F48BZF, and (G and H) hERG-E50BZF either before (black traces) or after 15 s of UV irradiation (blue traces). UV light was applied when channels were held in closed state at −100 mV or in the open state at 0 mV, as indicated. Scatter plot depicting time to half-maximum current before (black symbols, 0 s) and after UV irradiation (blue symbols, 15 s) after UV irradiation at a voltage of (I) −100 mV or (J) 0 mV. Scale bar represents 0.2 nA and 200 ms. n = 3, ns denotes p > 0.05, ^∗ denotes p ≤ 0.05, ^∗∗ denotes p ≤ 0.01, ^∗∗∗ denotes p ≤ 0.001.

Figure 6

Schematic of the effect UV irradiation on hERG-BZF channels. Schematic of hERG-BZF channels (A) in the open state and (B) in the closed state before UV irradiation. Black arrows depict transitions between open and closed conformations due to change in membrane voltage. Schematic of hERG-BZF channels after UV irradiation and cross-linking (C) in the open state and (D) in the closed state. Cross-links (red dashed lines) depict the PAS domain alpha A helix cross-linked to either the PAS domain or to the CNBHD. Robust black arrow depicts faster closing, and faint black arrow depicts slower opening.