Figure 1
From: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression
Mutations in the hERG PAS domain produce a range of expression defects. (a) Domain structure of hERG1a. Indicated are: Per-Arnt-Sim (PAS) domain, cyclic nucleotide binding domain (CNBD), transmembrane helices (S1-S6), extracellular turret (T), intramembrane pore (P) and engineered extracellular HA-epitope tag. Location of ER-retained mutations (G601S and F805C)4,37 and PAS-mutations employed in this study are indicated in red and green, respectively. (b) Structural model of the hERG PAS domain (yellow) and CNBD (cyan). PAS domain mutations described here shown in red. hERG cryoEM structure (PDB 5va1) described previously44. (c) Mutations reduce the expression of complex-glycosylated mature hERG channels. WT, PAS-mutant and severely misfolded (G601S and F805C) hERG stably expressed in HeLa cells and detected by immunoblotting. Immature core-glycosylated (~135 kDa) and mature complex-glycosylated (~155 kDa) hERG indicated with empty and solid arrows, respectively. GAPDH: loading control. Representative immunoblots shown (uncropped images in Supplementary Fig. S12). Solid line: different parts of the same gel. White space: separate gels. (d) Quantitative analysis of PAS-mutants expression defect. Mature hERG protein levels and PM-expression were determined by immunoblotting and PM-ELISA, respectively. Expression was normalized to hERG mRNA quantity and expressed as percent of WT. (e) Correlation between mature protein and PM hERG expression. Correlation determined by linear regression (R2 = 0.95). (f) PAS-mutants accumulate in intracellular compartments. PM and cellular hERG immunostained prior to or following fixation and permeabilization. WT-hERG shows strong PM distribution (white arrow) while select PAS mutants (F29L and T65P) are predominantly confined to intracellular compartments. Higher-magnification images and analysis of an additional mutant (M124R) shown in Supplementary Fig. S2. Scale bar: 10 µm, (g) Rescue of hERG folding restores PM expression. PM expression of WT and PAS-mutant hERG (F29L, C64Y, T65P and M124R) determined by PM-ELISA following low-temperature incubation (30 °C, 24 h) or treatment with the hERG pharmacochaperone E4031 (10 µM, overnight). Cell-surface expression normalized to mRNA abundance and expressed as percentage relative to untreated WT-hERG. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See Methods for explanation of statistical analysis).
