14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
Figure 8
Reversal of the 14-3-3θ suppression of rEag1 K+ currents by the 14-3-3 antagonist difopein.
(A) GST pull-down assay of the cell lysates prepared from HEK293T cells over-expressing the YFP vector, YFP-difopein, or YFP-R18 mutant. Pull-down products were detected by immunoblotting with the anti-14-3-3θ antibody. Compared to the vector control (lane 1), introduction of difopein (lane 2) resulted in a 75% and 64% reduction in the amount of 14-3-3θ pull-down by the GST-C0 and GST-N207 fusion proteins, respectively. In contrast, no significant difference was observed in the presence of the inactive mutant control (lane 3). (B) Normalized mean K+ current density recorded from HEK293 cells stably expressing rEag1 channels. As indicated, these stable cell lines were subject to transient transfection with various cDNA constructs. The mean current density at +40 mV for each co-expression condition was normalized with respect to that of the co-expression of rEag1 and difopein. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1-difopein co-expression control (*, t-test: p<0.05).