Trafficking assay of LQT2 mutants located within β9-strand.

<p>(A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after proteinase K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V_0.5 (open symbols) and 3 s isochronal deactivation V_0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate <i>P</i><0.05 (ANOVA) compared to WT. Bottom panel: Summary of the V_0.5 of steady-state inactivation for WT, N861H and N861I (same colours as in top panel). Mean data for all mutants are summarized in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077032#pone.0077032.s001" target="_blank">File S1</a>.