F-box protein-32 down-regulates small-conductance calcium-activated potassium channel 2 in diabetic mouse atria

Abstract

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation, but the underlying ionic mechanism for this association remains unclear. We recently reported that expression of the small-conductance calcium-activated potassium channel 2 (SK2, encoded by KCCN2) in atria from diabetic mice is significantly down-regulated, resulting in reduced SK currents in atrial myocytes from these mice. We also reported that the level of SK2 mRNA expression is not reduced in DM atria but that the ubiquitin-proteasome system (UPS), a major mechanism of intracellular protein degradation, is activated in vascular smooth muscle cells in DM. This suggests a possible role of the UPS in reduced SK currents. To test this possibility, we examined the role of the UPS in atrial SK2 down-regulation in DM. We found that a muscle-specific E3 ligase, F-box protein 32 (FBXO-32, also called atrogin-1), was significantly up-regulated in diabetic mouse atria. Enhanced FBXO-32 expression in atrial cells significantly reduced SK2 protein expression, and siRNA-mediated FBXO-32 knockdown increased SK2 protein expression. Furthermore, co-transfection of SK2 with FBXO-32 complementary DNA in HEK293 cells significantly reduced SK2 expression, whereas co-transfection with atrogin-1ΔF complementary DNA (a nonfunctional FBXO-32 variant in which the F-box domain is deleted) did not have any effects on SK2. These results indicate that FBXO-32 contributes to SK2 down-regulation and that the F-box domain is essential for FBXO-32 function. In conclusion, DM-induced SK2 channel down-regulation appears to be due to an FBXO-32-dependent increase in UPS-mediated SK2 protein degradation.

Keywords: HL-1 cells; SK channels; atrial fibrillation; atrogin-1; cardiomyocyte; cardiovascular disease; diabetes; diabetes mellitus; potassium channel; protein degradation; ubiquitin ligase.

Figure 1.

Atrogin-1 regulates SK2 protein expression in HL-1 cells. A, adenovirus-mediated overexpression of atrogin-1 in HL-1 cells was associated with a significant reduction in SK2 protein levels (*, p < 0.05; n = 3) but had no effect on those of SK1 and SK3 (p = N.S., n = 3). B, knockdown of atrogin-1 using atrogin-1 siRNAs achieved a 71% reduction in atrogin-1 protein expression in HL-1 cells, and this was accompanied by a significant 2.1-fold up-regulation of SK2 protein (*, p < 0.05; n = 3). Atrogin-1 knockdown had no significant effect on the levels of SK1 and SK3 protein expression (p = N.S., n = 3).

Figure 2.

SK channel expression in control and diabetic mouse atria. A, immunoblots of SK channels and atrogin-1 using homogenates of control and type 1 (streptozotocin-induced) diabetic mouse atria. Group data show significant down-regulation of SK2 and up-regulation of atrogin-1 protein expression in type 1 DM (n = 6 for each group; *, p < 0.05). B, immunoblots of SK channels and atrogin-1 using homogenates from control and type 2 db/db diabetic mouse atria. Group data show significant downregulation of SK2 and up-regulation of atrogin-1 protein expression in type 2 DM (n = 5 for each group; *, p < 0.05).

Figure 3.

Role of the ubiquitin-proteasome system in the regulation of SK2 protein expression. A, incubation with a proteasomal inhibitor, MG132 (24 h, 10 μm), abolished the effects of atrogin-1 on SK2 protein degradation (n = 6 for each group; *, p < 0.05). B, left panel, adenovirus-mediated up-regulation of atrogin-1 resulted in significant downregulation of SK2 in HL-1 cells cultured in HG (n = 6 for each group; *, p < 0.05). Right panel, knockdown of atrogin-1 with siRNA resulted in significant up-regulation of SK2 in HL-1 cells cultured in HG (n = 3 for each group; *, p < 0.05).

Figure 4.

Mutations in the PDZ-binding motif of SK2 prevented atrogin-1-mediated-ubiquitination and degradation of SK2. A, left panel, there was no alteration in atrogin-1ΔF protein expression compared with that of the WT atrogin-1. Right panel, co-transfection of atrogin-1 and FLAG-SK2 resulted in significant down-regulation of SK2 protein expression compared with the control (FLAG-SK2 co-transfected with empty plasmids) (*, p < 0.05, n = 3). However, co-transfection with atrogin-1ΔF had no effect on the level of SK2 expression compared with control (p = N.S., n = 3). B, top panel, immunoprecipitation with anti-ubiquitin antibody against HEK293 cell lysates with each combination of co-transfection. First lane, SK2 WT co-transfected with ubiquitin only; second lane, SK2 WT co-transfected with ubiquitin and atrogin-1; third lane, the SK2 PDZ binding motif mutant co-transfected with atrogin-1 and ubiquitin; fourth lane, SK2 WT co-transfected with atrogin-1ΔF and ubiquitin; fifth lane, lysate control; sixth lane, nonimmune IgG negative control. Ubiquitination of SK2 WT (second lane) was more substantially enhanced than all other combinations and controls. Nonimmune IgG control on the lysate of the second lane was negative (sixth lane). The experiment was independently repeated three times and produced similar results (shown in C). Bottom panel, the inputs of the tested samples as indicated in the top panel. SK2 and its mutant expression were detected by anti-FLAG antibody against the FLAG tag on the channels, and atrogin-1 and its mutant were detected by anti-myc antibody to the Myc tag fused with atrogin-1 and its mutant. C, group data for the experiments in B presented in dot scatterplots showing that significant SK2 ubiquitination occurred when SK2 WT and atrogin-1 WT were present (n = 3, p < 0.05). The positions of the lanes correspond to the position and composition of table columns in B. Data represent all anti-FLAG-reactive bands in densitometry arbitrary units (AU).

Figure 5.

Down-regulation of SK current densities in HEK293 cells by atrogin-1 co-transfection. SK2 WT cDNAs were transfected into HEK293 cells with atrogin-1 WT or atrogin-1ΔF cDNAs. Forty-eight hours after transfection, the apamin-sensitive currents were elicited from a holding potential of −60 mV to different testing potentials of −80 mV to +40 mV in +10-mV increments in the presence and absence of apamin (100 pm). The current–voltage relationships showed a significant reduction in apamin-sensitive K⁺ components (defined as SK2 currents in picoamperes/picofarads) in cells with co-expression of atrogin-1 WT compared with those with atrogin-1ΔF expression (n = 4 for both groups; *, p < 0.05).