Transfer RNA-mediated restoration of potassium current and electrical correction in premature termination long-QT syndrome hERG mutants

Figures

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  Figure 1. Engineered transfer RNAs (tRNAs) restore full-length and fluorescence of mutant GFP with PTC (A) Structure of GFP (PDB: 1GFL). Full-length GFP is in green and GFP-UAG (Y151X) is overlaid in blue. (B and C) Native mass spectrometry showing deconvolved masses for GFP and GFP N150X. (D) Flow cytometric analysis of GFP fluorescence where each cell is a single dot. (E) Engineered nonsense mutation in GFP (GFP-UAG) abolishes GFP fluorescence. (F and G) Stop codon suppression with Gln-tRNA partially rescues GFP fluorescence. (H–K) GFP fluorescence of GFP-UGA is also rescued (as shown in H) by (I) Arg-tRNA, (J) Gly-tRNA, and (K) Trp-tRNA and (G) with diminishing efficiencies. (L) Bar graph summarizes population data of normalized GFP fluorescence compared with WT (geometric mean, n = 3).

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  Figure 2. Rescue of patient hERG nonsense mutations with engineered transfer RNA (tRNA) in HEK293 cells (A) Illustration of engineered WT hERG with mCherry fluorescence tag on the N terminus, YFP on the C terminus, and BTX-647 tagging the extracellular BTX binding site. (B) Confocal images of HEK293 transiently transfected and labeled with BTX-647. Exemplar images show expression (left), full translation of hERG (middle), and trafficking of the channel to the membrane (left). Scale bar, 10 μm. (C) Flow cytometric analysis of population distribution for mCherry, YFP, and BTX-647 fluorescence, where each cell is represented by a single point in a trial. YFP is plotted as a function of mCherry to show the relative proportion of channels fully translated (left). BTX-647 is plotted as a function of mCherry to show relative fraction of hERG trafficked to the plasma membrane (right). (D) Cartoon depiction of hERG with nonsense mutation Q702X. Translation terminates before YFP (top), which is rescued with Gln-tRNA (bottom). (E) Exemplar confocal images show that Q702X mutants are expressed with mCherry fluorescence (top left) and are not fully translated, thus missing YFP fluorescence (top…

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  Figure 3. Mutant hERG co-expressed with an engineered transfer RNA (tRNA) is functionally similar to WT channels (A) Cartoon depiction of WT hERG transfected into HEK293. (B) Exemplar traces of WT hERG with a voltage-step protocol where the cell is depolarized from −80 mV to a step voltage ranging from −60 to +40 mV in 20 mV increments for 2 s and then back to +40 mV for 1 s with a repetition interval of 2 s. (C) HEK293 cells were transfected with the Q702X mutant alone (top) or with Gln-tRNA (bottom). (D) Exemplar current traces for Q702X voltage-step protocol showing that mutant channels to not exhibit much outward current (top). Gln-tRNA rescues potassium currents to levels comparable to those of WT (bottom). (E and F) R863X mutants also show reduced potassium currents, which are restored with Arg-tRNA. Format as in (C) and (D). (G and H) W1001X mutants show less reduction in potassium currents, and the addition of Trp-tRNA does not appear to markedly increase current size. Format as in (C) and (D). (I) Violin plots of population data for each of the conditions, where each point represents a single cell. Dunnett’s multiple comparisons test was used to determine statistical significance…

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  Figure 4. Engineered transfer RNA (tRNA) rescued prolonged action potentials in aGPVMs expressing mutant hERG (A) Action potentials were measured from isolated aGPVMs. (B) Confocal images of uninfected aGPVMs. Exemplar images show no mCherry (left) or YFP (right) expression. Scale bar, 50 μm. (C) Exemplar trace of action potentials measured from uninfected aGPVMs. (D) Cartoon depiction of adenovirus delivering WT hERG construct to aGPVM. (E) Confocal images show robust expression of mCherry (left) and YFP (right) for WT hERG. (F) Action potential of aGPVMs infected with WT hERG shows not much difference compared with uninfected aGPVMs. (G) Cartoon depiction of adenovirus delivering mutant R863X hERG (top) and rescue with another adenovirus carrying Arg-tRNA (bottom). (H) Confocal images show mCherry expression for both mutant (top left) and rescued hERG (bottom left). YFP is not expressed in mutant hERG (top right) but is rescued with Arg-tRNA (bottom right). (I) R863X-infected aGPVMs show prolonged action potentials (top), which is rescued with Arg-tRNA expression (bottom). (J) Violin plot of population data for each of the viral conditions, where each point represents average APD80…