Figure 5

Peripheral quality control engagement is dependent on conformational destabilization. (a) Mature hERG is destabilized at elevated temperature. Metabolic stability of mature WT, PAS-mutant (F29L and T65P) or temperature-rescued G601S (48 h at 26 °C, rG601S) hERG evaluated at 37 °C or 41 °C by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). Representative immunoblots shown (uncropped images in Supplementary Fig. S12). Solid line: different parts of the same gel. White space: separate gels. (b) Turnover kinetics of mature WT and F29L hERG fit using single-exponential decay functions. Similar results obtained for T65P and rG601S hERG (Supplementary Fig. S5). (c) Turnover rate-constants determined by curve fitting as in (b) and expressed as fold increase relative to 37 °C. (d) Pharmacological correction of hERG folding restores cell-surface stability. PM-turnover of WT and select PAS-mutants hERG measured by cell-surface ELISA following overnight (16 h) E4031 treatment (10 µM). (e) Pharmacochaperone treatment improves folding of nascent hERG at the ER but does not promote refolding of mature channels at the PM. Internalization of WT and select PAS-mutant hERG measured by PM-ELISA following acute (1 h) or overnight (16 h) E4031 pre-treatment (10 µM). (f) Delivery of PM-labelled T65P hERG to LAMP1-positive compartments evaluated by LCFM following 3 h chase. Lysosomal delivery is prevented by overnight pre-treatment with E4031 (10 µM). Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 2 µm, right) images shown. Magnified area indicated by white box. Analysis of WT and additional PAS-mutants in Supplementary Fig. S6. (g) Pharmacochaperone pre-treatment prevents endo-lysosomal trafficking of T65P hERG. Representative histogram of T65P hERG vesicular pH following 3 h chase. Overlay of multi-Gaussian peak-fits (mean ± SD) shown. N indicates total number of vesicles evaluated. (h) Mean luminal pH of hERG-containing endocytic vesicles measured by FRIA following overnight treatment with E4031 (10 µM) and 3 h chase at 37 °C. (i) Subset of temperature-rescued PAS-mutants are resistant to unfolding at physiological temperature. Internalization of WT and PAS-mutant hERG measured by PM-ELISA following low-temperature rescue (30 °C for 24 h) and unfolding (37 °C for 2 h). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See Methods for explanation of statistical analysis).