The mutation L69P in the PAS domain of the hERG potassium channel results in LQTS by trafficking deficiency

Figures

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  Figure 1.. (a): 12-lead electrocardiogram of the patient revealing a prolonged corrected QT interval of 478 ms. Heart frequency: 63/min; PQ interval: 140 ms; QRS duration: 80 ms; QT: 468 ms; P-duration: 110 ms; RR/PP interval: 952/950 ms. (b) Electrocardiogram monitoring of the patient showed torsades de pointes.

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  Figure 2.. Structure of PAS domain in hERG channel and impact of L69P mutation. (a) Multiple sequence alignment of PAS domain ERG channels from different organisms. The part of the domain including L69 (red) in hERG is highly conserved. (b) Final snapshots of MD simulation of wt and L69P mutant. The regions boxed in the structures are magnified. The structure of the PAS domain is shown as a gray cartoon with residue 69 as red sticks. (c) DSSP plot of secondary structural elements in wt and L69P mutant for residues 50 to 90 in PAS domain. The 3-helix formed by residues 67–69 in the wt structure vanishes in the mutant; dark red band indicated by the arrow. (d) Root mean square fluctuation (RMSF) calculation for wt (red) and L69P mutant (blue).

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  Figure 3.. Electrical properties of hERG-wt and L69 mutants. (a) Voltage protocol (top) and representative currents recorded from HEK293 cells expressing hERG-wt (a), mutant hERG-L69P (b) or from mock transfected cell (c). (B) Steady-state I/V relations of currents from A; symbols in I/V relation cross-reference with symbols on current traces. Inset: Mean steady-state currents at +20 mV from mock transfected cells and cells expressing wt or L69P mutant (n = number of cells). (C) Magnification of tail currents from HEK293 cell expressing L69P mutant (top) and hERG-wt (bottom). Exemplary tail currents were measured at −40 mV after pre-pulse to +40 mV (red) or −40 mV (black). (d) Mean activation curves from tail currents of hERG-wt or L69P mutant. Data were fitted with a Boltzmann function: I/Imax = 1/(1+ exp((V1/2-V)/k)) (eqn. 1), where V1/2 is the half maximum activation voltage and k the slope factor. Fitting of the data yields mean values for half maximal activation (V1/2) of −4 mV for the wt (open bar) and −7 mV for the mutant (black bar). The slope factor is 4 for the wt and mutant. (e) Mean tail currents (±SD) from cells expressing hERG-wt, different mutations of residue L69 and from…

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  Figure 4.. Impact of L69P mutation on protein synthesis and plasma membrane targeting. (a) Representative HEK292 cells expressing GFP tagged hERG-wt (top) or L69P mutant (bottom). Cells are show from left-to-right as bright field (left) and fluorescent images (central). The areas indicated by boxes are magnified in the right column. Arrows indicate the position of the plasma membrane. (b) Confocal image of isolated plasma membrane patches of HEK293 cells, which transiently express either the GFP tagged hERG wt (left column) or the L69P mutant (right column). The red channel shows fluorescently staining of membrane patches with CellMaskTM Deep Red, while the green channel reports fluorescence from GFP. The two lower rows show the red and green fluorescence of the respective membrane patches in a side view perspective. Note that the cells are fully decapitated leaving only isolated membrane patches. For a quantitative analysis, the fluorescence was measured from the side view images in regions of the red fluorescence and the respective area of the green channel. (c) Mean relative GFP fluorescence in membrane patches from HEK293 cells expressing hERG-wt or L69P mutant at 37°C or 27°C. Data…

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  Figure 5.. Flow cytometric analysis of surface expression with L69P mutants ± WT hERG channels. (a) Flow cytometry dot plot showing surface (BTX647 fluorescence) and total (YFP fluorescence) hERG expression in cells expressing BBS-hERG-YFP. Vertical and horizontal lines represent thresholds for YFP and BTX647-positive cells, respectively, based on analyses of single-color controls. Represented are YFP-positive cells with BTX647 signal above (red dots) or below threshold (green dots); BTX647-positive cells with YFP signal below threshold (gray dots); and untransfected cells (black dots). (b) Cumulative distribution histograms of Alexa647 fluorescence from flow cytometry analyses. Plot generated from population of YFP-positive cells (n ≥5000 cells per experiment). Dotted line is the threshold value for BTX647 signal. (c, d) Same experiments as in (a,b), but upon co-expression with WT hERG-CFP channels.