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First published online 17 February 2009
doi: 10.1242/jcs.036632

Journal of Cell Science 122, 775-779 (2009)
Published by The Company of Biologists 2009
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SUMOylation regulates Kv2.1 and modulates pancreatic β-cell excitability

Xiao-Qing Dai1,2, Jelena Kolic1,2, Paolo Marchi1, Simonetta Sipione1 and Patrick E. MacDonald1,2,*

1 Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2E1 Canada
2 The Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, T6G 2E1 Canada


Figure 1
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  		Fig. 1. SUMOylation regulates Kv2.1 and native INS-1 Kv current. (A) HEK 293 cells were transfected with the indicated cDNAs. Immunoprecipitation was performed using an anti-Kv2.1 antibody and analyzed by immunoblotting using anti-Kv2.1 and anti-GFP antibodies as indicated, and identified by the green and red signals, respectively, in the colored panel. Colocalization of these is shown in yellow. (B,C) Representative current traces and current-voltage relationships from HEK 293 cells expressing Kv2.1 alone (control, black circles) or together with SUMO1-YFP (SUMO1, black triangles), SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (white circles). (D) Current-voltage relationships from HEK 293 cells expressing Kv2.1 and infused with recombinant GST (black circles), SUMO1 (black triangles) or SUMO1+Ubc9 (open triangles) peptides. (E,F) Representative current traces and current-voltage relationships from INS1 832/13 cells expressing EGFP (control, black circles), SUMO1-YFP (SUMO1, black triangles), SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (open circles). *P<0.05, **P<0.01 and ***P<0.001 compared with control; error bars, s.e.m.

 

Figure 2
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  		Fig. 2. SUMOylation regulates native human β-cell Kv current. (A) Representative current traces from human β-cells expressing EGFP alone (control) or SUMO1-YFP (SUMO1) with Ubc9 and/or SENP1 as indicated. (B-D) Current-voltage relationships were from β-cells from three human donors expressing EGFP (black circles), SUMO1-YFP (black triangles), SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (open circles). (E) Same as B-D, but data from all donors was pooled. (F,G) Representative current traces and current-voltage relationships from human β-cells (two donors) infused with recombinant GST (black circles), SUMO1 (black triangles), or SUMO1+Ubc9 (open triangles). *P<0.05, **P<0.01 and ***P<0.001 compared with control; error bars, s.e.m.

 

Figure 3
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  		Fig. 3. SUMOylation regulates inactivation and recovery of Kv2.1 channels. (A) Representative current traces from HEK 293 cells expressing Kv2.1 alone (control) or with SUMO1-YFP (SUMO1), Ubc9 and/or SENP1 as indicated and normalized to peak current. (B) Inactivation time constants determined by fitting a single exponential decay function to the currents in panel A. (C) Recovery from inactivation following a 5-second depolarization to +30 mV in HEK 293 cells expressing Kv2.1 alone (black circles) or with SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (white circles). The Kv2.1+SUMO1-YFP curve was between the control and SENP1 curves and the Ubc9 curve (not shown). (D,E) The percent recovery to initial maximum current at 65.5 seconds and the recovery time constant determined by fitting the recovery curve to a single exponential function. (F) Voltage-dependence of steady-state inactivation of Kv2.1 expressed in HEK 293 cells either alone (black circles), with SUMO1-YFP (black triangles), with SUMO1-YFP+Ubc9 (white triangles) or with SUMO1-YFP+SENP1 (white circles). *P<0.05 and **P<0.01 compared with control; error bars, s.e.m.

 

Figure 4
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  		Fig. 4. SUMOylation regulates the excitability of INS1 832/13 β-cells. (A) Representative action potential traces induced by injection of a 10 pA current in INS1 832/13 cells expressing EGFP (control) or SUMO1-YFP (SUMO1), Ubc9 and/or SENP1 as indicated. (B-D) Action potential firing frequency, half-width and peak amplitudes for the groups shown in panel A. The half-width and peak amplitudes for the cells expressing SUMO1-YFP+Ubc9 were not analyzed because action potentials were rarely observed in this group. *P<0.05, **P<0.01 and ***P<0.001 compared with control; error bars, s.e.m.

 

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