Qi XY, Diness JG, Brundel BJ, et al. by Western blotting. Ca2+\activated K+ current (= .778, n = 5 for each). The surface expression of the SK2 protein from HEK293 cells cotransfected with JP2 and SK2 plasmids significantly increased by 1.02 0.14% (n = 5 for each) compared with the cells transfected with the SK2 channel alone (0.13 0.05%) ( .05, Figure ?Physique4B,4B, C). The results indicated that JP2 regulates the targeting of SK2 channel around the membrane. Open in a ELN484228 separate window Physique 4 JP2 increases SK2 channel surface expression in HEK293 cells. A, Western blot analysis showing the total expression of the SK2 protein ELN484228 from HEK293 cells cotransfected with JP2 and SK2 plasmids and with the cells transfected with the SK2 channel alone. B, The surface expression of the SK2 protein from HEK293 cells stably expressing SK2 and SK2+JP2 with cell surface biotinylation assay. Signals were corrected to total channel protein input and normalized. C, The bar graphs show no changes in the total expression of the SK2 protein (input), but the surface expression of the SK2 protein (biotinylated protein) from HEK293 cells cotransfected with JP2 and SK2 plasmids significantly increased compared with the cells transfected with the SK2 channel alone (* .05, n=5 per group) 2.5. Knockdown of JP2 depresses Ca2+\activated K+ currents in cardiac myocytes To expand our understanding of the physiological significance of JP2 binding to the SK2 channel in cardiomyocytes, we examined whether JP2 specifically modulates the SK2 channel function using an RNA interference technique. A small interference RNA oligonucleotide probe against a specific cDNA sequence of JP2 was found to be highly effective in knocking down of JP2 expression in the cardiac myocytes (observe Supplementary Information). Western blotting analysis revealed that the level of JP2 expression in the adult mouse cardiac tissue transduced with Ad\mediated siRNA specifically targeted against JP2 for 7 days was reduced to 47% and 48%, respectively, of the levels observed in the non\infected adult cardiac tissue (Ctrl\NT) and infected scramble siRNA (Ad\NC) cardiac tissue (= .0047 and = .0098, n = 5 per group from 5 hearts, Figure ?Physique5C,5C, D). No significant switch in expression levels of the SK2 channel was examined in the adult cardiac tissue infected with Ad\siJP2 compared with the controls (n = 5 ELN484228 per group from 5 hearts, Physique ?Physique5C,5C, E). Open in a separate window Physique 5 siRNA knockdown of JP2 depresses .05 with respect to Ad\NT or Ctrl\NC groups (n = 5 per group). E, The bar graphs show no changes in the expression of SK proteins in siRNA\infected adult mouse myocardium compared with Ad\NT or Ctrl\NC groups (n = 5 per group). F, Representative Rabbit Polyclonal to SLC25A6 traces of .01, ? .01, * .05 vs control siRNA groups, n = 8 per group). F, The bar graphs show reduced .05, ? ELN484228 .01 vs control ELN484228 siRNA groups (n = 8 for each group) We further investigated whether siRNA knockdown of JP2 may interfere with the SK2 channel function. The Ca2+\activated K+ current ( .05, ? .01, respectively, n = 8 for Ad\NC; n = 9 for Ad\siJP2). At ?120, ?110, +50 and +60 mV, the = .01, n = 8 for Ad\control; n = 9 for Ad\shRNA) as shown in Physique ?Figure5H.5H. Altogether, knockdown of JP2 led to a decrease in the = .92) (Physique ?(Figure5B).5B). siRNA knockdown of JP2 significantly decreased the amplitude.