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First published online 1 November 2005
doi: 10.1242/jcs.02635

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Downregulation of the HERG (KCNH2) K⁺ channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation

Hugh Chapman^1,*, Cia Ramström^1,2,*, Laura Korhonen³, Mika Laine^1,4, Kenneth T. Wann⁵, Dan Lindholm^1,3, Michael Pasternack¹ and Kid Törnquist^1,2,

¹ Minerva Foundation Institute for Medical Research, Biomedicum Helsinki, Haartmaninkatu 8, FI-00290, Helsinki, Finland
² Department of Biology, Åbo Akademi University, Artillerigatan 6, FI-20520 Turku, Finland
³ Department of Neuroscience, Unit of Neurobiology, Uppsala University, BMC, Box 587, SE-75123 Uppsala, Sweden
⁴ Department of Cardiology, Helsinki University Central Hospital, Stenbäckinkatu 9, FI-00290 Helsinki, Finland
⁵ Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF1 3XF, UK

View larger version (31K): [in a new window]   Fig. 1. The effect of ceramide on IHERG density in stably expressing HEK293 cells. (A) Whole-cell current recordings of IHERG in the absence and presence of 10 µM ceramide or dihydro-ceramide. From a holding potential of –80 mV cells were stepped to potentials between –70 mV and +40 mV for 2 seconds with tail currents observed on repolarisation to –60mV for 4.5 seconds. Inset, Identification of IHERG by the use of the inhibitor cisapride (1 µM) with the current evoked by a depolarising step to 0 mV. (B,C) Voltage-dependence of IHERG density with IHERG measured at the end of the depolarising steps (B) and as peak tail current (C). , control, n=12; , 10 µM ceramide, n=9. Significant differences in current density were observed (*P<0.05, ** P<0.001) compared to levels in the control. (D) Instantaneous IHERG density-voltage plot. Insets show the effect of 10 µM ceramide on the instantaneous IHERG density at 0 mV and the voltage protocol used, which consisted of a 2 second activating step to +40 mV, a 13 msecond step to –100 mV to relieve inactivation and then a step to potentials between +50 mV and –20 mV. IHERG was measured immediately following the end of the brief hyperpolarising step (n=7 for both control () and 10 µM ceramide (); *P<0.05). (E) The effect of ceramide concentration on the instantaneous IHERG density at 0 mV (voltage protocol as in D). The number of cells tested are given in parentheses (*P<0.05 compared to control current).

View larger version (14K): [in a new window]   Fig. 2. Time course of IHERG decline induced by 10 µM ceramide. (A) Typical recording obtained from a cell in control solution and then after 1 hour exposure to 10 µM ceramide. Tail currents at –60 mV were recorded following depolarisation to +40 mV for 2 seconds from the holding potential of –80 mV. The protocol was repeated every 12 seconds. (B) Plot of change in IHERG density from baseline against time. A baseline was established during 5-10 minutes of perfusion with control solution and then solutions were changed to 10 µM ceramide () or control (). Protocol as in A with IHERG taken as the peak tail current. The number of cells tested are given in parenthesis (*P<0.05, **P<0.001).

View larger version (27K): [in a new window]   Fig. 3. Effect of ceramide on HERG protein expression. (A) HERG protein expression was analysed by western blot. Ceramide-treated (10 µM for 60 minutes) cells were lysed and ultracentrifuged as described in Materials and Methods and proteins were separated by 6% SDS-PAGE. HERG protein was detected with monoclonal rabbit anti-HERG (1:1000). The higher molecular weight band (155 kDa) corresponds to the fully glycosylated mature HERG protein and the lower molecular weight band (135 kDa) corresponds to the core-glycosylated immature HERG protein. Results of the densitometric analysis are shown in the lower panel. The results represent the mean±s.e.m. of nine separate experiments. (B) Specific cell surface expression of HERG protein was analysed by biotin labelling. After incubation with ceramide cell surface proteins were biotinylated with a biotinylating reagent, sulfo-NHS-SS-biotin. HERG protein was immunoprecipitated and the biotinylated HERG channels were detected by horseradish peroxidase conjugated streptavidin. The blot shown is a representative of three separate experiments.

View larger version (52K): [in a new window]   Fig. 4. Effect of ceramide on HERG cell surface expression. (A) Ceramide-induced internalisation of HERG channels in HEK293 cells. In quantitative analysis of anti-HERG immunoreactivity statistically significant changes in plasma membrane and cytosolic immunofluorescence levels were observed after 30 minutes of ceramide treatment. Results are shown relative to nuclear fluorescence levels at baseline. Each bar gives the mean±s.e.m. of seven separate experiments. (B) Confocal sections of HEK cells labelled with anti-HERG antibody. Ceramide treatment for 60 minutes induced a clear internalisation of immunoreactivity from plasma membrane to cytosol. This internalisation was inhibited by the lysosomal inhibitor bafilomycin and by low-temperature treatment (16°C, 45 minutes), which curtails endocytosis. In contrast, the ceramide-induced internalisation was not inhibited by the proteosomal inhibitor MG132. The figures shown are representative cells of three to ten separate experiments. Bar, 10 µM.

View larger version (34K): [in a new window]   Fig. 5. Ubiquitylation of the HERG channel in HEK293 cells. (A) HERG-expressing HEK293 cells were exposed to ceramide 10 µM (60 minutes) and the HERG protein was immunoprecipitated. The amount of ubiquitin in HERG protein was determined by western blotting using an anti-ubiquitin antibody (clone P4G7) recognising both poly- and monoubiquitylated proteins. The same blot was also stained for HERG showing the levels of HERG protein in the cell lysates. The intensity of the bands was analysed and the bars show the ratio of ubiquitin and HERG in control conditions and in cells stimulated with 10 µM ceramide for 1 hour. The normalised values represent the mean±s.e.m. of five experiments. (B) Prior to treatment with 10 µM ceramide (60 minutes), HERG-expressing HEK293 cells were exposed for 60 minutes to either 20 µM MG132 or 0.25 µM bafilomycin. HERG protein was immunoprecipitated and the amount of ubiquitin was determined by western blot using an anti-ubiquitin antibody (clone P4G7). The bar graphs show the intensity of the bands as a ratio of ubiquitin and HERG. The normalised values represent the mean±s.e.m. of three experiments. (C) Downregulation of HERG through the lysosomal pathway. Control cells and ceramide-treated cells (10 µM for 60 minutes) were co-stained with antibodies for the lysosomal marker Lamp-1 (red) and HERG protein (green). The colocalisation is seen as yellow. The figures shown are representative cells of three separate experiments. Bar, 10 µM.

View larger version (19K): [in a new window]   Fig. 6. Effect of ceramide on HERG channel ubiquitylation and degradation in HEK cells. As shown in this study, ceramide leads to HERG channel ubiquitylation and subsequent degradation via lysosomes. However, ceramide also inhibits to some extent the basal turnover of HERG protein through proteosomes. Bafilomycin is a lysosome inhibitor whereas MG132 and lactacystin inhibit proteosomal degradation. See Discussion for further details.