Disruption of protein quality control of the human ether-à-go-go related gene K+ channel results in profound long QT syndrome

Abstract

Background: Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)-encoded K⁺ channel (K_v11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERG_T613M) and RNF207 frameshift (RNF207_G603fs) mutations. However, the underlying mechanistic underpinning remains unknown.

Objective: The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K⁺ channel subunits.

Methods: Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207.

Results: Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERG_T613M proteins for degradation. RNF207_G603fs exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERG_T613M subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERG_A614V, a known dominant-negative mutant subunit. Finally, the presence of RNF207_G603fs with hERG_T613M results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell-derived cardiomyocytes.

Conclusion: Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K⁺ channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.

Keywords: Cardiac ion channels; E3 ubiquitin ligase; Endoplasmic reticulum-associated degradation; Human ether a-go-go related gene (hERG)–encoded potassium channels; Human induced pluripotent stem cell-derived cardiomyocytes; Human induced pluripotent stem cells; Long QT syndrome; Protein quality control; RING finger protein 207 (RNF207).

Figure 1

A Dominant-negative effect and altered channel gating kinetics by hERG_T613M^. A: Amino acid sequence alignments for hERG_WT and hERG_T613M (residues 596–630) and RNF207_WT and RNF207_G603fs (residue 596 to the end of the polypeptide). The conserved selectivity filter of the hERG subunit GFG is underlined. The asterisk indicates mutated residues (613 in the hERG sequence and 603 in the RNF207 sequence). B: Representative recordings from HEK 293 cells expressing hERGWT alone, hERGWT:hERGT613M, hERGT613M alone, or nontransfected cells. C: Summary data of current densities for hERGWT alone (black traces) compared with hERG_WT:hERG_T613M (blue traces). n = 20–30 cells; *P < .05. D: Summary data for voltage-dependent activation using the peak tail current density fitted using the Boltzmann function (see Online Supplemental Table 1). E–H: Time constants of inactivation, recovery from inactivation, activation, and deactivation, respectively, from hERG_WT alone (black traces) compared with hERG_WT:hERG_T613M (blue traces). The insets show voltage-clamp protocols and representative current traces. n = 6–11 for inactivation; n = 9–11 for recovery from inactivation; n = 3 for activation; n = 5–10 for deactivation. *P < .05, **P < .01. Data shown are mean ± SEM. Analyses were performed using the Student t test. Expanded current traces in insets in panels E–H are shown in Online Supplemental Figure 3. ANOVA = analysis of variance; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; I = current; ms = millisecond; mV = millivolt; pA/pF = picoampere per picofarad; RNF207 = ring finger protein 207; s = second; SEM = standard error of the mean; V = voltage; WT = wild-type.

Figure 2

Colocalization of hERG and RNF207 in guinea pig ventricular cardiomyocytes. A: Confocal images showing colocalization among α-actinin2, hERG K⁺ channel subunits, and RNF207. Scale bar = 10 μm. The right panels show the corresponding fluorescence intensity profiles perpendicular to the z lines. B: Proximity ligation assay (PLA) for α-actinin2, hERG K⁺ channel subunits, and RNF207. C: Quantification of PLA signals per cell area (puncta/μm²). n = 15, 11, 15, 10, 9, and 9 cells from left to right bars; *P < .05. D: Auto-ubiquitinylation assay for RNF207_WT (lane 3, right) vs negative control (lane 1, left) and MDM2, a known E3 ubiquitin ligase (positive control, lane 2). Transfected HEK 293 cells were immunoprecipitated (IP) for RNF207-FLAG. An auto-ubiquitinylation assay was conducted on isolated protein, followed by SDS-PAGE and Western blot analysis (IB). Proteins were incubated in the presence of E1 and E2 ubiquitin enzymes, ubiquitin, and ATP. E: Ubiquitination assays. Ubiquitinated proteins were absent in the negative control (lane 1). The E3-ubiquitin band appeared for hERG_T613M incubated with RNF207_WT (lane 3), but not in the presence of RNF207_G603fs (lane 5) or with hERG_WT subunits (either with RNF207_WT [lane 2] or with RNF207_G603fs [lane 4]). F: Quantification of the data from panel E. n = 5 independent experiments for each group; *P < .05. Data shown are mean ± SEM. Analyses were performed using 1-way analysis of variance (ANOVA) with Brown-Forsythe post hoc analyses. AU = arbitraty units; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; RNF207 = ring finger protein 207; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM = standard error of the mean; Ub = ubiquitin; WT = wild-type.

Figure 3

Surface membrane and cytosolic expression of hERG K⁺ channel subunits after coexpression with RNF207_WT vs RNF207_G603fs. A: Immunofluorescence confocal microscopic imaging of HEK 293 cells transfected with different combinations of hERG_WT, hERG_T613M, RNF207_WT, and RNF207_G603fs. NP = nonpermeabilized; P = permeabilized. Scale bar = 10 μm). The last panel in each row represents a higher magnification image from the merged image as outlined in a red box. B: Schematic diagrams of hERG_WT-HA and hERG_T613M-Myc fusion constructs. C: Summary data of the fluorescence ratios (555 nm/633 nm). n = 6–10 cells; *P < .05, **P < .01. Data shown are mean ± SEM. Analyses were performed using ANOVA with Tukey’s post hoc analyses. ANOVA = analysis of variance; DAPI = 4’6-diamidino-2-phenylindole; GFP = green fluorescent protein; HA = hemagglutinin; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; RNF207 = ring finger protein 207; SEM = standard error of the mean; WT = wild-type.

Figure 4

Distinct effects of RNF207_WT vs RNF207_G603fs on the degradation of hERG K⁺ channel subunits. A: Degradation assay for HEK 293 cells collected before (time = 0) vs 3, 6, 12, and 24 hours after treatment. Cells were treated with brefeldin A (left panel), brefeldin A + MG 132 (middle panel), or brefeldin A + bafilomycin A1 (Baf A1; right panel). B–D: Summary data from panel A. *P < .05 in panel B. Data shown are mean ± SEM. n = 5 different independent experiments; *P < .05. Analyses were performed using ANOVA with Tukey’s post hoc analyses. ANOVA = analysis of variance; Baf A1 = bafilomycin A1; BFA = brefeldin A; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; IB = immunoblot; kDa = kilodaltons; RNF207 = ring finger protein 207; SEM = standard error of the mean; WT = wild-type.

Figure 5

Multiprotein complexes formed by hERG K⁺ channel subunits and RNF207. A: Transfected (T_x) HEK 293 cells were immunoprecipitated (IP) using anti-hERG antibody, and Western blot analysis (IB) was performed using anti-hERG and anti-FLAG antibodies to target the RNF207-FLAG fusion protein. Western blot analyses from lysates and IP are shown in lanes 1–5 and lanes 6–10, respectively. The negative control using IgG for IP is shown in lane 11. B: Schematic of hERG_WT ΔN, hERG_WT ΔC, hERG N-terminus, and hERG C-terminus constructs (tagged with HA). C: Coimmunoprecipitation of hERG-HA fragments that were cotransfected (Tx) in HEK 293 cells with RNF207_WT-FLAG. Immunoprecipitation (IP) followed by immunoblotting (IB) was performed using anti-HA (IP:HA, upper panel) and anti-FLAG (IB:FLAG, upper panel) antibodies, respectively. The reverse experiments were conducted as shown in the lower panel. Lanes 1–4 are lysate samples (20 μg each), and lanes 5–8 show immunoprecipitated samples transfected with hERG_WT ΔN, hERG_WT ΔC, hERG N-terminus, hERG C-terminus, and IgG negative control. Some nonspecific bands are seen in lanes 5–8 in the lower panel. The blue arrow in lane 5 in the lower panel shows a band of the expected size for hERG_WT ΔN. D: Rosetta model of hERG (from top to bottom): (1) view from the extracellular side of the membrane, (2) view from the transmembrane side, and (3) view from the intracellular side of the membrane. The backbone is shown in ribbon representation and colored by the rainbow color scheme from the N terminus (blue) to the C terminus (red). The side chain of the T613 residue in each subunit is shown using a space filling representation. ANOVA = analysis of variance; cyt = cytosolic; HA = hemagglutinin; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; IB = immunoblot; IP = immunoprecipitation; kDa = kilodaltons; RNF207 = ring finger protein 207; SEM = standard error of the mean; Tx = transfection; WT = wild-type.

Figure 6

Differential effects of RNF207_WT vs RNF207_G603fs on the hERG current. A: Representative whole-cell voltage-clamp recordings. B: Summary data of current density for hERG_WT:RNF207_WT (black traces) compared with hERG_WT:hERG_T613M:RNF207_WT (red traces) and hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs (blue traces). n = 14–19.C: Summary data for voltage-dependent activation using the peak tail current density fitted using the Boltzmann function (see Online Supplemental Table 1). D and E: Time constants of inactivation and recovery from inactivation for hERG_WT:RNF207_WT (black traces) compared with hERG_WT:hERG_T613M:RNF207_WT (red traces) and hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs (blue traces). F and G: Time constants for activation and deactivation, where closed Δ, □, and O represent the fast component and open Δ, □, and O represent the slow component of deactivation. n = 5–10 for inactivation; n = 6–12 for recovery from inactivation; n = 3–5 for activation; n = 5–10 for deactivation. *P < .05, **P < .01. In panels B and C, **P < .01 for hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs compared with hERG_WT:RNF207_WT and hERG_WT:hERG_T613M:RNF207_WT throughout positive voltages and was shown only at the end of the curves for clarity. Data shown are mean ± SEM. Analyses were performed using ANOVA with Tukey’s post hoc analyses. ANOVA = analysis of variance; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; RNF207 = ring finger protein 207; SEM = standard error of the mean; WT = wild-type.

Figure 7

Regulation of APDs of hiPSC-CMs by RNF207. A: Representative action potential recordings (iCell, Cellular Dynamics) in cells expressing hERG_WT:RNF207_WT (black trace), hERG_WT:hERG_T613M:RNF207_WT (red trace), and hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs (blue trace) as well as a nontransfected cell (gray trace). B–F: Summary data for action potential recordings in nontransfected cells (labeled “Non-TF”; gray bar) compared with hERG_WT:RNF207_WT (labeled “WT”; black bar), hERG_WT:hERG_T613M:RNF207_WT (labeled “Rescue”; red bar), and hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs (labeled “Mutant”; blue bar) at baseline (solid bars) vs 1 μM E-4031 (striped bars). Data are shown for average diastolic potential (panel B), peak action potential (panel C), action potential amplitude (panel D), action potential duration at 50% repolarization or APD₅₀ (panel E), and action potential duration at 90% repolarization or APD₉₀ (panel F). Data shown represents the average of 5 action potentials per cell, with n = 6–9 cells for baseline recordings and n = 3–5 cells for E-4031 recordings. *P<.05, **P<.01, §P<.001. Data shown are mean ± SEM. Analyses were performed using ANOVA with Tukey’s post hoc analyses. ANOVA = analysis of variance; APD = action potential duration; APD50 and APD90 = APD at 50% and 90% repolarization; hERG = human ether-à-go-go related gene; hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocytes; non-TF = nontransfected cells; RNF207 = ring finger protein 207; SEM = standard error of the mean; WT = wild-type.

Figure 8

Regulation of hERG currents by RNF207 in hiPSC-CMs and a schematic diagram of RNF207 interaction with hERG-encoded K⁺ channels in adult ventricular myocytes. A: Representative E-4031–sensitive currents recorded from hiPSC-CMs expressing hERG_WT:RNF207_WT (black traces), hERG_WT:hERG_T613M:RNF207_WT (red traces), and hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs (blue traces). B: Summary data of current density for the 3 groups of cells. n = 5–6. C: Summary data for voltage-dependent activation using the peak tail current density fitted using the Boltzmann function (see Online Supplemental Table 1). n = 5–6. In panel B, *P < .05 for hERG_WT:RNF207_WT compared with hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs throughout positive voltages and was shown only at the end of the curve for clarity. In panel C, *P < .05 for hERG_WT:RNF207_WT and hERG_WT:hERG_T613M:RNF207_WT compared with hERG_WT:hERG_T613M:RNF207_WT:RNF207_G603fs throughout positive voltages and was shown only at the end of the curves for clarity. Analyses were performed using ANOVA with Tukey’s post hoc analyses. D: Schematic diagram of RNF207 interaction with hERG-encoded K⁺ channels (K_v11.1) with trafficking and degradation pathways (generated using BioRender, Toronto, Canada). ANOVA 5 analysis of variance; hERG = human ether-à-go-go related gene; hiPSC-CM 5 human induced pluripotent stem cell-derived cardiomyocytes; I = current; RNF207 = ring finger protein 207; SEM = standard error of the mean; V = voltage; WT = wild-type.