14-3-3h also exhibited similar punctate immunofluorescence staining. We also compared the subcellular distribution of 14-3-3h and rEag1 by performing the subcellular fractionation experiment. Via sucrose gradient centrifugation, the P2 fraction of rat forebrain homogenates was separated into multiple fractions, from which we collected the synaptosomal (SPM) fraction. As illustrated in the left panel of Figure 6A, both 14-3-3h and rEag1 proteins were present in the SPM fraction, implying a co-localization of the two proteins in the synapse. Extractions with Triton X-100 further divided the SPM fraction into the postsynaptic density I (PSD I; one Triton X- 100 wash) and the PSD II (two Triton X-100 washes) fractions, which provide insightful information on the subcellular localiza- tion of synapse-related proteins: the postsynaptic marker PSD-95 was highly enriched in both the PSD I and the PSD II fractions, whereas the presynaptic marker synaptophysin was only present in the PSD I fraction (Fig. 6A, right panel). Consistent with the aforementioned punctate co-localization patterns in cultured hippocampal neurons (see Fig. 5B), we found that rEag1 as well as 14-3-3h were detected in both the PSD I and the PSD II fractions (Fig. 6A, right panel). Figure 6B provides a quantitative summary of the relative abundance of each protein in different subcellular fractions. Consistent with the co-immunoprecipitation result (see Fig. 4A), rEag1, but not rEag2, displays a similar synaptic localization profile to that of 14-3-3h. Altogether these data provide strong evidence in support of the association of 14-3- 3h with rEag1 in the brain. Suppression of rEag1 K+ currents by the over-expression of 14-3-3h If the preceding inference on the protein-protein interaction is true, then is it possible that 14-3-3h may affect the functional property of rEag1 K+ channels? To address this question, we studied the functional expression of rEag1 channels in the absence or presence of the over-expression of 14-3-3h. Figure 7A exemplifies the representative result observed in HEK293T cells: over-expression of 14-3-3h led to about 30% reduction of the amplitude of rEag1 K+ currents. Similarly, in Xenopus oocytes, over-expression of 14-3-3h resulted in more than 50% reduction of rEag1 K+ currents (Fig. 7B). By contrast, the functional expression of rEag1 channels was not significantly affected by the over- expression of the auxiliary b1 subunit of Kv channels (Kvb1) (Fig. 7B), a protein similar in size with 14-3-3h. Other than the suppression of current amplitude, co-expression with 14-3-3h failed to exert discernible effect on the gating properties (such as steady-state voltage dependence and gating kinetics) of rEag1 channels (Fig. 7C). Difopein (dimeric fourteen-three-three peptide inhibitor) is a high-affinity 14-3-3 antagonist that contains two R18 peptides connected by a linker and competitively binds to all isoforms of 14- 3-3, thereby effectively disrupting 14-3-3/ligand interaction in cells [32]. On the other hand, a monomeric R18 mutant in which two key residues are replaced by lysine fails to bind to 14-3-3 and hence serves as an inactive mutant control of difopein [32]. We also applied difopein and the R18 mutant to verify the functional effect of 14-3-3h on rEag1. Figure 8A shows that the cell lysates prepared from HEK293T cells over-expressing difopein, but not the R18 mutant, significantly attenuated the amount of 14-3-3h precipitated by both GST-C0 and GST-N207 fusion proteins, demonstrating that difopein effectively interfered with the in- teraction between 14-3-3h and the N- and C-termini of rEag1. Importantly, when we co-expressed 14-3-3h with difopein or the R18 mutant in HEK293 cells stably transfected with rEag1, the suppression effect of 14-3-3h on rEag1 K+ currents was abolished 14-3-3h Interacts with rEag1 in the presence of difopein, but not the R18 mutant (Fig. 8B), supporting the idea that 14-3-3h binding is indeed indispensable for the foregoing functional modulation of rEag1 channels. To further understand the mechanism of 14-3-3h-induced suppression of rEag1 K+ currents, we asked whether co-expression with 14-3-3h could affect the protein expression or membrane trafficking of rEag1 channels. As shown by the surface biotinyla- tion data in Figure 9, in HEK293T cells, neither the total protein density nor the surface expression efficiency of rEag1 channels was significantly altered by the over-expression of 14-3-3h, suggesting that the suppression effect of 14-3-3h cannot be attributed to an alteration of the biosynthetic process of rEag1 protein. An alternative interpretation of the functional effect of 14-3-3h is that the molecule may reduce the single channel conductance of rEag1 K+ channels. To test this hypothesis, we performed non- stationary fluctuation analysis on macroscopic currents to compare rEag1 single channel conductance under different co-expression paradigms. Figure 10 shows that despite about 30% reduction in the macroscopic current density, co-expression with 14-3-3h failed to significantly affect the estimated single channel conductance of rEag1 K+ channels. Discussion The 14-3-3 protein family has been shown to physically interact with a wide variety of different proteins (over a hundred), including enzymes, cytoskeletal and structural proteins, kinases, membrane proteins, and transcription factors; 14-3-3 proteins may thus mediate diverse regulatory functions in many cellular processes, such as apoptosis, cell cycle, protein trafficking, and signal transduction [33,34]. 14-3-3 proteins are also involved in the regulation of membrane ion channels [29,35,36]. The mechanism underlying these diverse aspects of channel regulation by 14-3-3 proteins remains elusive, as the physiological outcome of 14-3-3 modulation seems to be determined in a 14-3-3 isoform- and binding partner-specific manner. Moreover, the detailed structural basis of protein-interacting domain, as well as 14-3-3 isoform specificity, is lacking. The results from our GST pull-down analyses indicate that the interaction with 14-3-3h is mediated by the cytoplasmic N- and C- terminal regions of rEag1 proteins. Specifically, we propose that the N-terminal 14-3-3h interaction site is mainly located at the PAS domain. The PAS domain is a small, modular domain found in numerous signaling molecules in both prokaryotes and eukaryotes [25]. Some of the well characterized PAS domain- dependent signaling pathways include transcriptional regulations induced by xenobiotic compounds (e.g., dioxin), circadian rhythm (e.g., CLOCK protein), or hypoxia response (e.g., hypoxia inducible factor, HIF); in most cases, the PAS core serves as the protein-protein interaction domain and may therefore determine the specificity of interactions among different signaling molecules [25,37,38]. Although PAS domains are found in all members of the EAG K+ channel family, until now their role in voltage-gated ion channels remains unclear. The current study offers a new perspective on this issue: the PAS domain may serve as a modular structural motif through which 14-3-3 proteins, and perhaps some other 14-3-3-binding proteins as well, may regulate the function of the EAG K+ channel family. In addition, we propose that the C-terminal 14-3-3h interaction site of rEag1 comprises the CNBHD, which is a conserved structural domain for the EAG K+ channel family, as well as cyclic nucleotide-gated and hyperpolarization-activated cyclic nucleo- tide-modulated channels [1,26,39]. The functional significance of CNBHD in the EAG K+ channel family is obscure, since cyclic PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e41203