14-3-3h Interacts with rEag1 (*, t-test: p,0.05). (B) (Left panel) Representative K+ currents recorded from oocytes expressing rEag1 in the absence or presence of 14-3-3h. The molar ratio for cRNA co-injection was 1:5 and 1:10 for 14-3-3h and Kvb1, respectively. The pulse protocol was identical to that described in (A). (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of 14-3-3h. (C) Biophysical properties of rEag1 channels in the absence (open circles) or presence (filled diamonds) of 14-3-3h. The voltage-dependant curves for steady-state activation (upper left panel), activation kinetics (upper right panel), deactivation kinetics (lower left panel), and non-superimposable Cole-Moore shift (lower right panel) were analyzed as described previously [17]. Data were collected from recordings performed in oocytes. doi:10.1371/journal.pone.0041203.g007 mammals, there are seven 14-3-3 isoforms: a/b, e, g, c, t/h, f/d, and s [20]. Except for the s isoform, 14-3-3 proteins are abundantly expressed in the brain, and have been implicated in the modulation of neurotransmission, brain development, and learning and memory [21,22,23]. The potential interaction of 14-3-3h with the N-terminus of the rEag1 protein was further validated by our yeast two-hybrid assay result showing that yeasts co-transformed with 14-3-3h and the plasmid encoding the rEag1-N207 segment were capable of growing in synthetic leucine-lacking media (Fig. 1B). Furthermore, yeasts co-transformed with 14-3-3h and the plasmid encoding the cytoplasmic C-terminus (amino acids 493–962) of the rEag1 protein (rEag1-C0) were also growing in the leucine-lacking medium (Fig. 1B), suggesting that 14-3-3h may interact with the C-terminus of rEag1 as well. To address this hypothesis, we employed GST pull-down assay with GST fusion proteins encoding the N- or C-terminus of the rEag1 protein (GST-N207 or GST-C0). As depicted in Figure 1C, in vitro translated 14-3-3h was efficiently retained by both GST-N207 and GST-C0 fusion proteins, but not by the GST protein per se (Fig. 1C), indicating a direct interaction between 14-3-3h and the GST fusion proteins. We also performed GST pull-down assay with the cell lysates prepared from HEK293T cells transfected with myc-tagged 14-3- 3h (myc-14-3-3h). Figures 1D and 1E demonstrate that myc-14-3- 3h was proficiently precipitated by the fusion proteins GST-N207 and GST-C0, respectively, further supporting the idea that multiple 14-3-3h-interacting sites may exist within the rEag1 protein. Since there are seven isoforms in the 14-3-3 protein family and some of the 14-3-3-interacting proteins have been shown to display specific binding affinity with just a subset of the 14-3-3 isoforms [24], we next asked whether the rEag1 protein exhibited a preferential interaction with specific members of the 14-3-3 protein family. Except for the s isoform that is virtually absent in the brain, various 14-3-3 isoforms were cloned from the rat brain cDNA library and used for GST pull-down assay with the rEag1 GST fusion proteins GST-N207 and GST-C0. As illustrated in Figure 2, all but the e isoform were precipitated by both GST- N207 and GST-C0, with 14-3-3h showing a significantly higher binding efficiency. This observation strongly suggests that 14-3-3h is the preferred binding partner of both the N- and C-terminal rEag1 fusion proteins. To further address the structural basis of these novel interac- tions, we generated additional GST fusion proteins encoding specific protein domains of rEag1. The cytoplasmic N-terminus of rEag1 protein contains a Per-Arnt-Sim (PAS) (amino acids 14– 145) domain that constitutes parts of the common structural features for the EAG K+ channel family [25]. To test the hypothesis that the PAS domain may contribute to the interaction between 14-3-3h and the N-terminus of the rEag1 protein, we created another N-terminal fusion protein GST-N163 that encodes the complete PAS domain. Interestingly, Figure 3A shows Figure 8. Reversal of the 14-3-3h 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-14-3-3h antibody. Compared to the vector control (lane 1), introduction of difopein (lane 2) resulted in a 75% and 64% reduction in the amount of 14-3-3h 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). doi:10.1371/journal.pone.0041203.g008 PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41203