Sodium permeable and “hypersensitive” TREK‐1 channels cause ventricular tachycardia

Figure 1. Identification of a heterozygous KCNK2 (TREK‐1) mutation in a patient with RVOT‐VT A12‐lead ECG during exercise of the proband (10772‐3) presenting with LBBB (with inferior axis) tachycardia being typical for its origin from the RVOT.B, C(B) Electropherogram and nucleotide sequence of KCNK2 illustrating a heterozygous c.800T > C single nucleotide exchange with a predicted non‐synonymous amino acid exchange (p. Ile267Thr; shortly: I276T). Localization of the TREK‐1I267T residue (highlighted in blue) within a highly conserved signature sequence (red) upon partial sequence alignment with TREK‐1 orthologues. The I276T mutation is located in the second pore domain of the TREK‐1 channel (indicated in a cartoon in B), or in a pore homology model based on the crystal structure of TREK‐2 (C).DPrioritization scheme for filtering nucleotide variants obtained after WES.

Figure 2. TREK‐1I267T causes a dominant‐negative reduction in outward currents and depolarizes membrane potentials Representative voltage‐clamp measurements in Xenopus oocytes injected with wild‐type TREK‐1 (black) or TREK‐1I267T (blue) cRNA (1 ng/oocyte). Currents were recorded 48 h after cRNA injection, applying a ramp protocol, for 3.5 s rising from −120 mV to +40 mV, from a holding potential of −80 mV.Mean current amplitudes of TREK‐1‐ and TREK‐1I267T‐mediated currents analyzed at +40 mV. Numbers of experiments are indicated within the bar graph.Mean membrane potential (Em) of TREK‐1 (black) and TREK‐1I267T (blue). Numbers of experiments are indicated within the bar graph.Representative measurement of the current–voltage relationship of TREK‐1I267T.Representative current–voltage relationship measurements in oocytes injected with TREK‐1 cRNA (2.5 ng/oocyte), TREK‐1I267T (2.5 ng/oocyte), wild‐type 1.25 ng TREK‐1 (mimicking haploinsufficiency), or with wild‐type TREK‐1 and TREK‐1I267T cRNA (1.25 ng each/oocyte, mimicking a heterozygous state). Current–voltage relationship was recorded from a holding potential of −80 mV, applying 10 mV steps from −130 to +50 mV with a duration of 200…

Figure 3. The TREK‐1I267T mutation alters the potassium selectivity and introduces a sodium permeability A, B(A) Representative voltage‐clamp recordings of wild‐type TREK‐1 (black) and TREK‐1I267T (blue) using different extracellular K+ concentrations, by replacing extracellular NaCl to KCl, and (B) the respective analysis of the Erev of TREK‐1 (black) or TREK‐1I267T (blue) as a function of the external K+ concentration (n = 10). The slope of the TREK‐1I267T mutant was only 13.2 mV/decade.CAnalysis of the Erev of TREK‐1 (black), mutant TREK‐1I267T (blue), and TREK‐1 co‐expressed with TREK‐1I267T (dark blue) while gradually removing the extracellular Na+, by replacing NaCl to NMDG‐Cl (n = 12). The K+ concentration was maintained at 2 mM.DRepresentative current traces of TREK‐1 (black) and TREK‐1I267T (blue) recorded in a bath solution containing 96 mM Na+ or 0 Na+ (NaCl to NMDG‐Cl with Ko + maintained at 2 mM).EPercentage of current change when Na+ was removed from the extracellular recording solution, as in (D). Numbers of experiments are indicated within the bar graph.FAverage current amplitude of TREK‐1 or TREK‐1I267T in extracellular solution containing 98 mM K+ but no Na+ (all NaCl…

Figure 4. TREK‐1I267T speeds early repolarization and depolarizes the membrane potential of spontaneously beating HL‐1 cells Fluorescence imaging of HL‐1 cells transfected with EGFP‐tagged TREK‐1 or TREK‐1I267T.Action potential (AP) frequency, as beats per minute (bpm), counted in HL‐1 cells (HL‐1) bathed in Claycomb medium containing 100 μM norepinephrine or HL‐1 cells transfected with TREK‐1‐EGFP (WT) or TREK‐1I267T‐EGFP (I267T). The number of experiments are indicated in the bar graphs.Patch clamp measurements in the current‐clamp mode of HL‐1 cells transfected with TREK‐1‐EGFP or mutant TREK‐1I267T‐EGFP. Boxes indicate the zoom area for panel (E).Analyses of the AP duration, APD50, and APD90 of HL‐1 cells transfected with TREK‐1‐EGFP or TREK‐1I267T‐EGFP. The number of experiments are indicated in the bar graphs.Illustration of the hyperpolarization observed following an AP of HL‐1 cells transfected with TREK‐1‐EGFP or TREK‐1I267T‐EGFP.Analysis of the afterhyperpolarization (AHP) observed in HL‐1 cells transfected with TREK‐1‐EGFP (n = 5) or TREK‐1I267T‐EGFP (n = 6).Analyses of the maximum diastolic hyperpolarization (max. HP) of HL‐1 cells transfected with TREK‐1‐EGFP (n = 5) or…

Figure 5. TREK‐1I267T reduces the affinity of TREK‐1 channel blockers and activators, but TREK‐1I267T is rescued by the activator BL‐1249 Analysis of the Em of TREK‐1I267T channel‐mediated currents without drug application (n = 15, blue) and in the presence of different drugs. 80 μM fluoxetine (n = 8), 62 μM verapamil (n = 13), 50 μM 2‐APB (n = 4), 500 μM riluzole (n = 4), or 5 μM BL‐1249 (n = 8, red) were tested.Chemical structure of BL‐1249. Representative current trace of the TREK‐1I267T mutant recorded in ND96 solution (blue) and after application of 5 μM BL‐1249 (red). The arrows indicate the respective reversal potential.Fold increase in TREK‐1 and TREK‐1I267T channel‐mediated currents by 10 μM BL‐1249. Numbers of experiments are indicated within the bar graph.The lower panel shows the EC50 of BL‐1249. Native TREK‐1 EC50 = 12.8 ± 3.8 μM (n = 3); TREK‐1I267T EC50 = 15.2 ± 3.4 μM (n = 8). The upper panel shows the maximum fold increase derived from the Hill equation; TREK‐1: 35‐fold, TREK‐1I267T: 42.4‐fold.Em of TREK‐1I267T expressing oocytes depending on BL‐1249 and on the extracellular K+ concentration in the presence of BL‐1249.The Em of the TREK‐1I267T mutant in the presence of…

Figure 6. TREK‐1I267T has an increased stretch‐sensitivity, a slowed stretch‐desensitization, and increased arachidonic acid sensitivity Representative inside‐out macropatch measurements of TREK‐1 (black) and TREK‐1I267T (blue) at different negative pressures applied via the pipette.Normalized current at the steady state of desensitization plotted against the negative pressure applied for TREK‐1 (black) and TREK‐1I267T (blue) (n = 5).Representative macropatch recordings at −50 mm H2O column of TREK‐1 (black) and TREK‐1I267T (blue).Average time constants of desensitization (τdesens.) derived from the bi‐exponential fit of current traces at −50 mm H2O column; τfast (filled circles) and τslow (open circles) (n = 5).Relative amplitudes of the fast and the slow component of current desensitization and the non‐desensitizing persistent current for TREK‐1 and TREK‐1I267T (n = 5).Relative current activation of TREK‐1 (black) or TREK‐1I267T (blue) using different intracellular arachidonic acid (AA) concentrations. TREK‐1 (n = 7); TREK‐1I267T (n = 8).Data information: Data are presented as mean ± SEM. Data in (B) are analyzed either by unpaired Student's t‐test or by Welch's t‐test. Data in (D) are…

Figure 7. Altered β1‐adrenoreceptor modulation in TREK‐1I267T channels Voltage‐clamp measurement of wild‐type TREK‐1 co‐expressed with a β1‐adrenoreceptor in Xenopus oocytes before (black) and after (red) application of 1 μM norepinephrine. Currents were recorded from a holding potential of −80 mV applying a ramp protocol for 3.5 s rising from −120 mV to +40 mV. Illustrated are the currents averaged from six cells.Voltage‐clamp measurement of TREK‐1I267T co‐expressed with a β1‐adrenoreceptor in Xenopus oocytes before (black) and after (red) application of 1 μM norepinephrine. Illustrated are the currents averaged from seven cells.Analysis of the β1‐adrenoreceptor‐mediated inhibition of wild‐type TREK‐1 (black) and TREK‐1I267T (blue) at +40 mV by 1 μM norepinephrine. The number of experiments is indicated within the respective bars.Zoom in from (B) to hyperpolarized potentials, illustrating the increased inward currents of TREK‐1I267T after application of 1 μM norepinephrine (red).Quantification of the activation of inward currents by TREK‐1I267T at −90 mV by 1 μM norepinephrine. The number of experiments is indicated within the bar.Data information: Data are presented as mean ± SEM. Data…