α-mannosidase I-dependent N-linked glycosylation affects hERG channel functional availability during reactivation

Figures

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  Fig. 1. The long-term effect of inhibitors of hERG channel glycosylation on IhERG in HEK293 cells. (A) Representative IhERG traces of vehicle and long-term (48 h) application of different glycosylation inhibitors of hERG channel (tunicamycin 0.5 µg/mL; kifunensine 100 µM; Deoxymannojirimycin 10 mM) are shown. Pulse protocol is shown above. (B) The effect of glycosylation inhibitors on terminal IhERG recorded at the end of the test pulse of 4 s were plotted against the potential as shown as IhERG.term-V relationship. (C) The effect of glycosylation inhibitors on peak outward tail IhERG recorded at the beginning of the pulse at −40 mV were plotted against the potential as shown as IhERG.tail-V relationship. Data were expressed as mean ± SE. Tunicamycin (0.5 µg/mL) significantly decreased IhERG.term and IhERG.tail as shown (At the maximum tail current of +60 mV, # means p < 0.05). kifunensine (100 µM) not reduced IhERG.term but slightly reduced IhERG.tail as shown (At the maximum tail current of +60 mV, * means p = 0.042). Deoxymannojirimycin (10 mM) exhibited a similar inhibitory effect on IhERG.term and IhERG.tail as shown (At the maximum tail current of +60 mV, † means p = 0.013).

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  Fig. 2. Actions of α-mannosidase I-dependent N-linked glycosylation inhibitor on IhERG reactivation. (A) Representative IhERG traces (left) showing hERG current recovery from inactivation were elicited by a 30 ms test protocol from −150 mV to +60 mV in vehicle or kifunensine (100 µM) for 48 h. The recovery-from-inactivation relationship (right) displayed the relationship between amplitude of peak tail current and the 30 ms test pulse membrane potentials in vehicle and kifunensine. (B) Representative IhERG traces (left) showing hERG current recovery from inactivation were elicited by a 5 ms test protocol from −150 mV to +60 mV in vehicle or kifunensine (100 µM) for 48 h. The recovery-from-inactivation relationship (right) displayed the relationship between amplitude of peak tail current and the 5 ms test pulse membrane potentials in vehicle and kifunensine. Kifunensine significantly reduced IhERG.tail at the potential (−150 mV – −100 mV) as shown above (* means p < 0.05). (C) Normalized recovery-from-inactivation current relationship from the 5 ms test protocol fitted with a Boltzmann function in vehicle- and kifunensine-treated cells. (D, E) Relationship between the time constants…

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  Fig. 3. Actions of α-mannosidase I-dependent N-linked glycosylation inhibitor on IhERG inactivation. (A) Representative IhERG traces showing the effect of kifunensine (100 µM) on the inactivation of IhERG.tail for 48 h. A three-pulse protocol was used to measure the inactivation current as shown above. (B) The effect of kifunensine (100 µM) on the voltage-dependence of the time constant of inactivation fitted to a single exponential. Data were expressed as mean ± SE.

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  Fig. 4. Actions of α-mannosidase I-dependent N-linked glycosylation inhibitor on IhERG deactivation. (A) Representative IhERG traces elicited by pulse protocol shown above, which displays deactivation current in vehicle and 100 μM kifunensine for 48 h. (B) Relationship between the time constants of deactivation current and the membrane potentials in the absence (vehicle) and presence of kifunensine. Insets in panel B to show the ration of the fast component of the decay amplitude (Afast) to whole decay amplitude (Afast + Aslow). Data were expressed as mean ± SE.

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  Fig. 5. Actions of α-mannosidase I-dependent N-linked glycosylation inhibitor on IhERG activation. (A) Representative activating IhERG traces of vehicle and kifunensine (100 µM) were recorded by an envelope of tail test shown in above in hERG-HEK cells for 48 h. (B) The normalized amplitude of the tail IhERG was plotted against the pulse duration. Data were expressed as mean ± SE.

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  Fig. 6. Dose-dependent inhibition of reactivating IhERG by α-mannosidase I-dependent N-linked glycosylation inhibitor. (A) Representative reactivating IhERG in vehicle, 25 μM, 100 μM and 200 μM kifunensine applied for 48 h. Pulse protocol was identical to that shown in panel A inset. (B) Normalized reactivation current was plotted against the various concentration of kifunensine. Data were expressed as mean ± SE. * p < 0.05 vs. vehicle; # p < 0.05 vs. 20 µM kifunensine; † p < 0.05 vs. 25 µM kifunensine.

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  Fig. 7. Effect of α-mannosidase I-dependent N-linked glycosylation inhibitor on hERG protein expression. (A) Representative Western blot bands showing the 155-kDa mature glycosylated form and the 135-kDa core-glycosylated form of hERG in neonatal cardiomyocytes treated with vehicle or kifunensine at different concentrations for 48 h. β-actin was used as the loading control. (B, C) Quantification of the 155-kDa and 135-kDa hERG bands normalized to β-actin in neonatal cardiomyocytes. (D) Representative Western blot bands showing the 155-kDa and 135-kDa forms of hERG in hERG-HEK293 cells treated with vehicle or kifunensine at different concentrations for 48 h.

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  Fig. 8. A voltage-dependent three states model of hERG channels gating properties.