multiple-gaussian fits for herg vesicular ph  (OriginLab corp)

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: PAS-mutant hERGs are sorted for lysosomal delivery from the cell surface. ( a ) hERG is targeted to LAMP1-positive endo-lysosomal compartments. Endocytic WT, M124R and C64Y hERG pool labelled by Ab capture (15 min at 37 °C) and remaining cell-surface hERG blocked with unconjugated secondary F(ab′) 2 (1 h on ice). Cells then chased at 37 °C for 3 h prior to fixation. Lysosomal compartments labelled with LAMP1 pAb. hERG (green) and LAMP1 (magenta) staining visualized by LCFM. Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 5 µm, right) images shown. Magnified area indicated by white box. Analysis of additional mutants (F29L, R56Q, T65P) in Supplementary Fig. . ( b ) Representative distribution of vesicular pH for WT and T65P hERG containing endocytic vesicles following 3 h chase. Overlay of multi-Gaussian peak-fits shown and mean pH ± SD indicated. N indicates total number of vesicles analyzed in a representative experiment. ( c ) PAS-mutations accelerate hERG endo-lysosomal delivery kinetics. Mean luminal pH of vesicles containing WT or T65P hERG measured by FRIA. Anti-HA Ab and FITC-Fab were bound on ice and FRIA was performed after 1- to 6-h chase. ( d ) Mean luminal pH of vesicles containing WT and PAS-mutant hERG following 3 h chase. ( e , f ) Lysosomal activity contributes to degradation of mature hERG proteins. Metabolic stability of WT and PAS-mutants hERG evaluated by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). V-ATPase inhibition with Bafilomycin A1 (BafA1, 200 nM), or proteasome inhibition with Bortezomib (Bort, 3 µM) or Ixazomib (Ixa, 3 µM) attenuated the rapid degradation of PAS-mutants. Mature complex-glycosylated (~155 kDa) and ER-resident core-glycosylated (~135 kDa) hERG indicated by solid and empty arrows, respectively. Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See methods and materials for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Mutagenesis, Staining, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: 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. ). 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. ). ( 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. . ( 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).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Control, Mutagenesis, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay