Figure 3
From: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression
Trafficking of mature PAS-mutants at the PM confers rapid elimination. (a) PAS mutant channels are subject to accelerated turnover at the cell-surface. Removal of WT and select PAS-mutant hERG (F29L, R56Q, T65P and A78P) from the cell-surface was measured by PM-ELISA. Turnover kinetics were fit using single-exponential decay functions (solid curves). Similar results obtained for the rest of the PAS mutants (data not shown). (b) PM half-life of WT- and mutant hERG calculated from curve-fitting as in (a). (c) Time course of WT and PAS-mutant (F29L and T65P) internalization. The disappearance of hERG from the cell-surface during a 0–7 minute chase was measured by ELISA and expressed as percent of initial cell-surface hERG remaining. Internalization kinetics fit using single-exponential decay functions. (d) Amount of hERG internalized during a 5-minute interval measured by PM-ELISA and expressed as percent of initial cell-surface pool lost. (e) Endocytic recycling kinetics of WT, T65P and F29L hERG in HeLa cells. Endosomal hERG pool labelled by Ab capture (20 min at 37 °C); recycling to the PM was measured by sandwich ELISA (0–20 min at 37 °C). Amount of endosomal hERG recycled to the PM expressed as percent of the initial labelled endosomal hERG pool. (f) Endocytic recycling of WT and PAS-mutant hERG during a 10-minute chase, measured as in (e). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. indicates no significant difference (See Methods for explanation of statistical analysis).
