gms | German Medical Science

Question: Mutations in the HERG gene are associated with distinct consequences on potassium (IKr) currents demonstrated in heterologous expression systems. Typically mutations causing long QT syndrome (LQT-2) lead to disruption of channel synthesis or defective protein trafficking, whereas few mutations known to cause short-QT syndrome are gain of function mutations. We studied four HERG mutations and one putative polymorphism (R176W), identified in arrhythmia patients to elucidated the biochemical alterations on HERG protein and IKr currents.

Method used: We performed site-directed mutagenesis to introduce the four mutations (cis double mutant V409L_W410C, R752P, T865I, R883Q) into the wild-type HERG cDNA. Western blot analysis was used to analyze the glycosylation patterns. Immunostaining and confocal laser microscopy of transiently transfected COS-7 cells were used to localize the mutated HERG proteins in the different cell compartments. We also tested the effect of lower temperature (27°C) and E4031 on protein trafficking. To analyze currents, we expressed the mutant HERG cRNA in Xenopus oocytes (20-22°C).

Results: Western blot analysis showed mature glycosylation of all altered HERG proteins. Immunofluorescence demonstrated that mutants V409L_W410C, R752P and T865I were trafficking deficient. The regular trafficking of T865I at 37°C to the cell membrane however, could be achieved by incubation with E4031 or coexpression with the WT. The other mutants could be rescued at lower incubation temperatures. In Xenopus oocytes all mutants generated detectable IKr currents. The currents of R176W, R752P and the coexpression of WT with T865I resemble to the WT currents. The ramp currents of R883Q were significantly increased. The double mutant V409L_W410C leads to distinct changes in channel kinetics.

Conclusions: Mutations in HERG are associated with various in-vitro and in-vivo dysfunctions that not only include mechanisms with reduced IKr but also with increased IKr.